Method and apparatus for desorption and ionization of analytes

ABSTRACT

This invention relates generally to methods and apparatus for desorption and ionization of analytes for the purpose of subsequent scientific analysis by such methods, for example, as mass spectrometry or biosensors. More specifically, this invention relates to the field of mass spectrometry, especially to the type of matrix assisted laser desorption/ionization, time-of-flight mass spectrometry used to analyze macromolecules, such as proteins or biomolecules. Most specifically, this invention relates to the sample probe geometry, sample probe composition, and sample probe surface chemistries that enable the selective capture and desorption of analytes, including intact macromolecules, directly from the probe surface into the gas (vapor) phase without added chemical matrix.

BACKGROUND OF THE INVENTION

[0001] This invention relates generally to methods and apparatus fordesorption and ionization of analytes for the purpose of subsequentscientific analysis by such methods, for example, as mass spectrometry(MS) or biosensors. Generally, analysis by mass spectrometry involvesthe vaporization and ionization of a small sample of material, using ahigh energy source, such as a laser, including a laser beam. Thematerial is vaporized from the surface of a probe tip into the gas orvapor phase by the laser beam, and, in the process, some of theindividual molecules are ionized by the gain of a proton. The positivelycharged ionized molecules are then accelerated through a short highvoltage field and let fly (drift) into a high vacuum chamber, at the farend of which they strike a sensitive detector surface. Since thetime-of-flight is a function of the mass of the ionized molecule, theelapsed time between ionization and impact can be used to determine themolecule's mass which, in turn, can be used to identify the presence orabsence of known molecules of specific mass.

[0002] All known prior art procedures which present proteins or otherlarge biomolecules on a probe tip for laser desorption/ionizationtime-of-flight mass spectrometry (TOF) rely on the preparation of acrystalline solid mixture of the protein or other analyte molecule in alarge molar excess of acidic matrix material deposited on the baresurface of a metallic probe tip. (The sample probe tip typically ismetallic, either stainless steel, nickel plated material or platinum).Embedding the analyte in such a matrix was thought to be necessary inorder to prevent the destruction of analyte molecules by the laser beam.The laser beam strikes the solid mixture on the probe tip and its energyis used to vaporize a small portion of the matrix material along withsome of the embedded analyte molecules. Without the matrix, the analytemolecules are easily fragmented by the laser energy, so that the mass,and identity, of the original macromolecule is very difficult orimpossible to determine.

[0003] This prior art procedure has several limitations which haveprevented its adaptation to automated protein or other macrobiologicalmolecular analysis. First, in a very crude sample it is necessary topartially fractionate (or otherwise purify the sample as much aspossible) to eliminate the presence of excessive extraneous materials inthe matrix/analyte crystalline or solid mixture. The presence of largequantities of components may depress the ion signal (either desorption,ionization and/or detection) of the targeted analyte. Such purificationis time-consuming, expensive, typically results in low recovery (orcomplete loss) of the analyte, and would be very difficult to do in anautomated analyzer.

[0004] Second, while the amount of analyte material needed for analysisby the prior art method is not large (typically in a picomole range), insome circumstances, such as tests on pediatric patients, analyte fluidsare available only in extremely small volumes (microliters) and may beneeded for performing several different analyses. Therefore, even thesmall amount (i.e., volume) needed for preparation of the analyte/matrixcrystalline mixture for a single analysis may be significant. Also, onlya tiny fraction (a few thousandths or less) of analyte used in preparingthe solid analyte/matrix mixture for use on the probe tip is actuallyconsumed in the desorption or mass spectrometric analysis. Anyimprovement in the prior art procedure which would make it possibleto 1) use much less analyte, 2) to locate the analyte or multipleanalytes on the probe tip or surface in a predetermined location, 3) toperform repeated analyses of the same aliquot of analyte (e.g., beforeand after one or more chemical and or enzymatic reactions), and 4) toconduct the test in a more quantitative manner, would be highlyadvantageous in many clinical areas.

[0005] Third, the analyte protein, or other macromolecule, used inpreparing the solid solution of analyte/matrix for use on the probe tipis not suitable for any subsequent chemical tests or procedures becauseit is bound up (i.e., embedded) in the matrix material. Also, all of thematrix material used to date is strongly acidic, so that it wouldadversely affect many chemical reactions which might be attempted on themixture in order to modify the analyte molecules for subsequentexamination. Any improvement in the procedure which made it possible toconduct subsequent chemical modifications or reactions on the analytemolecules, without removing them from the matrix or the probe tip orwithout “matrix” altogether, would be of enormous benefit to researchersand clinicians.

[0006] The first successful molecular mass measurements of intactpeptides and small proteins (only up to about 15 kDa) by any form ofmass spectrometry were made by bombarding surfaces with high energyparticles (plasma desorption and fast atom bombardment massspectrometry); this breakthrough came in 1981 and 1982. Improvementscame in 1985 and 1986, however, yield (signal intensities), sensitivity,precision, and mass accuracy remained relatively low. Higher molecularmass proteins (about 20 to 25 kDa) were not observed except on rareoccasions; proteins representing average molecular weights(approximately 70 kDa) were not ever observed with these methods. Thus,evaluation of most proteins by mass spectrometry remains unrealized.

[0007] In 1988, Hillenkamp and his coworkers used UV laser desorptiontime-of-flight mass spectrometry and discovered that when proteins ofrelatively high molecular mass were deposited on the probe tip in thepresence of a very large molar excess of an acidic, UV absorbingchemical matrix (nicotinic acid) they could be desorbed in the intactstate. This new technique is called matrix-assisted laserdesorption/ionization (MALDI) time-of-flight mass spectrometry. Notethat laser desorption time-of-flight mass spectrometry (without thechemical matrix) had been around for some time, however, there waslittle or no success determining the molecular weights of large intactbiopolymers such as proteins and nucleic acids because they werefragmented (destroyed) upon desorption. Thus, prior to the introductionof a chemical matrix, laser desorption mass spectrometry was essentiallyuseless for the detection of specific changes in the mass of intactmacromolecules. Note that the random formation of matrix crystals andthe random inclusion of analyte molecules in the solid solution is priorart.

[0008] There are a number of problems and limitations with the prior artmethods. For example, previously, it has been found that it is difficultto wash away contaminants present in analyte or matrix. Other problemsinclude formation of analyte-salt ion adducts, less than optimumsolubility of analyte in matrix, unknown location and concentration ofanalyte molecules within the solid matrix, signal (molecular ion)suppression “poisoning” due to simultaneous presence of multiplecomponents, and selective analyte desorption/ionization. Priorinvestigators, including Karas and Hillenkamp have reported a variety oftechniques for analyte detection using mass spectroscopy, but thesetechniques suffered because of inherent limitations in sensitivity andselectivity of the techniques, specifically including limitations indetection of analytes in low volume, undifferentiated samples.(Hillenkamp, Bordeaux Mass Spectrometry Conference Report, pp. 354-62(1988); Karas and Hillenkamp, Bordeaux Mass Spectrometry ConferenceReport, pp. 416-17 (1988); Karas and Hillenkamp, Analytical Chemistry,60:2299 (1988); Karas, et al., Biomed. Environ. Mass Spectrum (inpress).) The use of laser beams in time-of-flight mass spectrometers isshown, for example, in U.S. Pat. Nos. 4,694,167; 4,686,366, 4,295,046,and 5,045,694, incorporated by reference.

[0009] The successful volatilization of high molecular weightbiopolymers, without fragmentation, has enabled a wide variety ofbiological macromolecules to be analyzed by mass spectrometry. Moreimportantly perhaps, it has illustrated the potential of using massspectrometry more creatively to solve problems routinely encountered inbiological research. Most recent attention has been focused on theutility of matrix-assisted laser desorption/ionization (MALDI)time-of-flight (TOF) mass spectrometry (MS), largely because it is rapid(min), sensitive (<pmol sample required), and permits complex mixturesto be analyzed.

[0010] Although MALDI-TOF MS continues to be useful for the staticdetermination/verification of mass for individual analytes, in the caseof biopolymers, it is often differences in mass that provide the mostimportant information about unknown structures. Thus, for routine use instructural biology, an unfortunate limitation of the MALDI-TOF MStechnique relates to sample preparation and presentation (deposition) onan inert probe element surface, specifically, the requirement thatanalytes be embedded (i.e., co-solidified) on the probe surface in afreshly prepared matrix of crystalline organic acid. The randomdistribution of analyte in a heterogeneous display of crystal matrix onthe probe element surface requires the deposition of far more analyte orsample than is needed for the laser desorption process, even for thecollection of more than adequate mass spectra (e.g., multiple sets of100 shots each). The remaining portion of the analyte is usually notrecovered for additional analyses or subsequent characterizations. Eventhough 1 to 10 pmol (sometimes less) of analyte are typically requiredfor deposition on the probe surface, it has been estimated that lessthan a few attomoles are consumed during laser desorption. Thus, only 1part in 10⁵ or 10⁶ of the applied analyte may be necessary; the rest islost.

[0011] Another important loss of potential data associated with theembedding of analyte in a solid matrix is the reduction or the completeelimination of ability to perform subsequent chemical and/or enzymaticmodifications to the embedded analyte (e.g., protein or DNA) remainingon the probe surface. Only another aliquot of analyte, or the ability torecover the embedded analyte free of matrix (difficult with lowrecovery), allows what we now refer to as differential mass spectrometryto be performed to derive structural data.

[0012] In addition, there has been limited application of MS inbiological fields, likely due to the fact that many biologists andclinicians are intimidated by MS and/or skeptical in regard to itsusefulness. Further, MS is perceived as inaccessible or too costly,particularly because SDS polyacrylamide gel electrophoresis is anadequate substitute in some instances where MALDI would be applied(e.g., separation of crude biological fluids). In addition, MALDI hashad little exposure in biological and clinical journals.

SUMMARY OF THE INVENTION

[0013] An object of the invention is to provide improved methods,materials composition and apparatus for coupled adsorption, desorptionand ionization of multiple or selected analytes into the gas (vapor)phase.

[0014] Another object is to provide a method and apparatus foraffinity-directed detection of analytes, including desorption andionization of analytes in which the analyte is not dispersed in a matrixsolution or crystalline structure but is presented within, on or abovean attached surface of energy absorbing “matrix” material throughmolecular recognition events, in a position where it is accessible andamenable to a wide variety of chemical, physical and biologicalmodification or recognition reactions.

[0015] Another object is to provide such a method and apparatus in whichthe analyte material is chemically bound or physically adhered to asubstrate forming a probe tip sample presenting surface.

[0016] A further object is to provide means for the modification ofsample presenting surfaces with energy-absorbing molecules to enable thesuccessful desorption of analyte molecules without the addition ofexogenous matrix molecules as in prior art.

[0017] A further object is to provide the appropriate density ofenergy-absorbing molecules bonded (covalently or noncovalently) in avariety of geometries such that mono layers and multiple layers ofattached energy-absorbing molecules are used to facilitate thedesorption of analyte molecules of varying masses.

[0018] A further object is to provide multiple combinations of surfacesmodified with energy-absorbing molecules, affinity-directed analytecapture devices, phototubes, etc.

[0019] An additional object is to provide such a method and apparatus inwhich the substrate forming the probe tip or other sample presentingsurface is derivatized with one or more affinity reagents (a variety ofdensities and degrees of amplification) for selective bonding withpredetermined analytes or classes of analytes.

[0020] A further object is to provide such a system in which theaffinity reagent chemically bonds or biologically adheres to the targetanalyte or class of analytes.

[0021] A still further object is to provide a method and apparatus fordesorption and ionization of analytes in which unused portion of theanalytes contained on the presenting surface remain chemicallyaccessible, so that a series of chemical, enzymatic or physicaltreatments of the analyte may be conducted, followed by sequentialanalyses of the modified analyte.

[0022] A further object is to provide a method and apparatus for thecombined chemical or enzymatic modifications of target analytes for thepurpose of elucidating primary, secondary, tertiary, or quaternarystructure of the analyte and its components.

[0023] Another object is to provide a method and apparatus fordesorption and ionization of analyte materials in which cations otherthan protons (H⁺) are utilized for ionization of analyte macromolecules.

[0024] Thus, in accomplishing the foregoing objects, there is providedin accordance with the present invention, an apparatus for measuring themass of an analyte molecule of an analyte sample by means of massspectrometry, said apparatus comprising a spectrometer tube; a vacuummeans for applying a vacuum to the interior of said tube; electricalpotential means within the tube for applying an accelerating electricalpotential to desorbed analyte molecules from said analyte sample; samplepresenting means removably insertable into said spectrometer tube, forpresenting said analyte sample in association with surface associatedmolecule for promoting desorption and ionization of said analytemolecules, wherein said surface molecule is selected from the groupconsisting of energy absorbing molecule, affinity capture device,photolabile attachment molecule and combination thereof; an analytesample deposited on said sample presenting means in association withsaid surface associated molecules, whereby at least a portion of saidanalyte molecules not consumed in said mass spectrometry analysis willremain accessible for subsequent chemical, biological or physicalanalytical procedures; laser beam means for producing a laser beamdirected to said analyte sample for imparting sufficient energy todesorb and ionize a portion of said analyte molecules from said analytesample; and detector means associated with said spectrometer tube fordetecting the impact of accelerated ionized analyte molecules thereon.

[0025] In addition, in accomplishing the foregoing objects, there isprovided in accordance with the present invention, a method in massspectrometry to measure the mass of an analyte molecule, said methodcomprising the steps of: derivitizing a sample presenting surface on aprobe tip face with an affinity capture device having means for bindingwith an analyte molecule; exposing said derivitized probe tip face to asource of said analyte molecule so as to bind said analyte moleculethereto; placing the derivitized probe tip with said analyte moleculesbound thereto into one end of a time-of-flight mass spectrometer andapplying a vacuum and an electric field to form an acceleratingpotential within the spectrometer; striking at least a portion of theanalyte molecules bound to said derivitized probe tip face within thespectrometer with one or more laser pulses in order to desorb ions ofsaid analyte molecules from said tip; detecting the mass of the ions bytheir time of flight within said mass spectrometer; and displaying suchdetected mass.

[0026] Further, in accomplishing the foregoing objects, there isprovided in accordance with the present invention, a method of measuringthe mass of analyte molecules by means of laser desorption/ionization,time-of-flight mass spectrometry in which an energy absorbing materialis used in conjunction with said analyte molecules for facilitatingdesorption and ionization of the analyte molecules, wherein theimprovement comprises presenting the analyte molecules on or above thesurface of the energy absorbing material, wherein at least a portion ofthe analyte molecules not desorbed in said mass spectrometry analysisremain chemically accessible for subsequent analytical procedures.

[0027] Additionally, in accomplishing the foregoing objects, there isprovided in accordance with the present invention, an apparatus forfacilitating desorption and ionization of analyte molecules, saidapparatus comprising: a sample presenting surface; and surfaceassociated molecules, wherein said surface associated molecules areselected from the group consisting of energy absorbing molecule,affinity capture device, photolabile attachment molecule and combinationthereof, said surface associated molecules associated with said samplepresenting surface and having means for binding with said analytemolecules.

[0028] Further, there is provided a method for capturing analytemolecules on a sample presenting surface and desorbing/ionizing saidcaptured analyte molecules from said sample presenting surface forsubsequent analysis, said method comprising: derivitizing said samplepresenting surface with an affinity capture device or photolabileattachment molecule having means for binding with said analytemolecules; exposing said derivitized sample present surface to a samplecontaining said analyte molecules; capturing said analyte molecules onsaid derivitized sample presenting surface by means of said affinitycapture device or photolabile attachment molecule; and exposing saidanalyte molecules, while bound to said derivitized sample presentingsurface by means of said affinity capture device or photolabileattachment molecule, to an energy or light source to desorb at least aportion of said analyte molecules from said surface.

[0029] Additionally, in accordance with the present invention, there isprovided a method for preparing a surface for presenting analytemolecules for analysis, said method comprising: providing a substrate onsaid surface for supporting said analyte; derivitizing said substratewith an affinity capture device or photolabile attachment moleculehaving means for selectively bonding with said analyte; and a means fordetecting said analyte molecules bonded with said affinity capturedevice or photolabile attachment molecule.

[0030] Further, in accomplishing the foregoing objects, there isprovided in accordance with the present invention, a sample probe forpromoting desorption of intact analytes into the gas phase comprising: asample presenting surface; and an energy absorbing molecule associatedwith said sample presenting surface, wherein said sample probe promotesdesorption of an intact analyte molecule positioned on, above or betweenthe energy absorbing molecules when said sample probe is impinged by anenergy source. Further, the energy absorbing molecule in the probe isselected from the group consisting of cinnamamide, cinnamyl bromide, 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid.

[0031] Additionally, in accomplishing the foregoing objects, there isprovided in accordance with the present invention, a sample probe fordesorption of intact analyte into the gas phase, comprising: a samplepresentation surface; and a surface associated molecule wherein saidsurface associated molecule is a photolabile attachment molecule havingat least two binding sites, wherein at least one site is bound to thesample presentation surface and at least one site is available to bindan analyte and wherein the analyte binding site is photolabile.

[0032] In addition, in accomplishing the foregoing objects there isprovided in accordance with the present invention, a sample probe forpromoting desorption of intact analytes into the gas phase comprising: asample presentation surface; and either

[0033] a mixture of at least two different molecules selected from thegroup consisting of an affinity capture device, an energy absorbingmolecule and a photolabile attachment molecule associated with saidsample presentation surface; wherein when an analyte is associated withsaid sample probe, said sample probe promotes the transition of theanalyte into the gas phase when said sample probe is impinged by anenergy source; or at least two different affinity capture devicesassociated with said sample presentation surface;

[0034] wherein, when said sample probe is impinged by an energy source,said sample probe promotes the transition of an analyte molecule intothe gas phase at different rates depending on the affinity capturedevice associated with said analyte molecule.

[0035] In addition, in accomplishing the foregoing objects there isprovided in accordance with the present invention, a sample probe forpromoting desorption of intact analyte into the gas phase, comprising: asample presentation surface; and either a surface associated molecule,wherein said surface associated molecule can function both as an energyabsorbing molecule and as an affinity capture device; or a surfaceassociated molecule wherein said surface associated molecule is aphotolabile attachment molecule having at least two binding sites,wherein at least one site is bound to the sample presentation surfaceand at least one site is available to bind an analyte and wherein theanalyte binding site is photolabile.

[0036] Additionally, there is provided in the present invention, amethod in mass spectrometry to measure the mass of an analyte molecule,said method comprising the steps of: derivitizing a sample presentingsurface on a probe tip face with a photolabile attachment molecule(PAM), wherein said PAM has at least two binding sites, one binding sitebinds to the sample presenting surface and at least one binding site isavailable for binding with an analyte molecule; exposing saidderivitized probe tip face to a source of said analyte molecule so as tobind said analyte molecule thereto; placing the derivitized probe tipwith said analyte molecules bound thereto into one end of atime-of-flight mass spectrometer and applying a vacuum and an electricfield to form an accelerating potential within the spectrometer;striking at least a portion of the analyte molecules bound to saidderivitized probe tip face within the spectrometer with one or morelaser pulses in order to desorb ions of said analyte molecules from saidtip; detecting the mass of the ions by their time of flight within saidmass spectrometer; and displaying such detected mass.

[0037] In addition, there is provided a method of measuring the mass ofanalyte molecules by means of laser desorption/ionization,time-of-flight mass spectrometry in which a photolabile attachmentmolecule (PAM) is used in conjunction with said analyte molecules forfacilitating desorption and ionization of the analyte molecules, theimprovement comprising: presenting the analyte molecules on or above thesurface of the PAM, wherein at least a portion of the analyte moleculesnot desorbed in said mass spectrometry analysis remain chemicallyaccessible for subsequent analytical procedures.

[0038] There is further provided in accordance with the presentinvention, a sample probe for promoting of differential desorption ofintact analyte into the gas phase, comprising: a sample presentationsurface; and at least two different photolabile attachment moleculesassociated with said sample presentation surface; wherein, when saidsample probe is impinged by an energy source, said sample probe promotesthe transition of an analyte molecule into the gas phase at differentrates depending on the photolabile attachment molecule associated withsaid analyte molecule.

[0039] Additionally, there is provided in accordance with the presentinvention, a sample probe for promoting desorption of intact analytesinto the gas phase comprising: a sample presenting surface; and aphotolabile attachment molecule associated with said sample presentingsurface; wherein, when said sample probe is impinged by an energysource, said sample probe promotes the transition of an intact analytemolecule into the gas phase.

[0040] Further, there is provided in accordance with the presentinvention, a method for biopolymer sequence determination comprising thesteps of: binding a biopolymer analyte to probe tip containing a samplepresenting surface having a surface selected molecule selected from thegroup consisting of an energy absorbing molecule, an affinity capturedevice, a photolabile attachment molecule and a combination thereof;desorption of biopolymer analyte in mass spectrometry analysis, whereinat least a portion of said biopolymer is not desorbed from the probetip; analyzing the results of the desorption modifying the biopolymeranalyte still bound to the probe tip; and repeating the desorption,analyzing and modifying steps until the biopolymer is sequenced.

[0041] Other and further objects, features and advantages will beapparent and the invention more readily understood from a reading of thefollowing specification and by reference to the accompanying drawingsforming a part thereof, wherein the examples of the presently preferredembodiments of the invention are given for the purposes of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The foregoing and other objects and advantages of the inventionwill be apparent from the following specification and from theaccompanying drawings.

[0043]FIG. 1A (upper profile) shows the mass spectrum of the threepeptides (human histidine rich glycoprotein metal-binding domains(GHHPH)₂G (1206 Da), (GHHPH)₅G (2904 Da), and human estrogen receptordimerization domain (D473-L525) (6168.4 Da)) desorbed in the presence ofneutralized energy absorbing molecules (sinapinic acid, pH 6.2). FIG. 1B(lower profile) shows the sequential in situ metal (Cu)-binding of thepeptides in the presence of neutral energy absorbing molecules.

[0044]FIG. 2A (top profile) shows the mass spectrum of the human caseinphosphopeptide (5P, 2488 Da) desorbed in the presence of neutralizedenergy absorbing molecules (sinapinic acid, pH 6.5). FIG. 2B (secondfrom top profile) shows the sequential in situ 5 min alkalinephosphatase digestion to remove phosphate groups from thephosphopeptide. FIG. 2C (third from top profile) shows the mass spectrumof the phosphopeptide after further in phosphatase digestion in thepresence of acidic energy absorbing molecules (2,5 dihydroxybenzoicacid, pH 2) as described in prior art.

[0045]FIG. 3 shows a composite mass spectra of the (GHHPH)₅G peptide(2904 Da) before (lower profile) and after (upper profile) in situdigestion by carboxypeptidase P in the presence of neutralized energyabsorbing molecules (sinapinic acid, pH 6.2).

[0046]FIG. 4 shows a composite matrix-assisted laser desorption massspectra of peptide mixtures desorbed from solid glass,polypropylene-coated stainless steel, polystyrene-coated stainless steeland solid nylon probe elements.

[0047] SEAC

[0048]FIG. 5, profile A shows the mass spectrum of sperm activatingfactor (933 Da) and neurotensin (1655 Da) (and their multipleNa-adducts) in the peptide solution unadsorbed by the IDA-Cu(II)surface. FIG. 5, profile B, shows the mass spectrum of angiotensin I(1296.5 Da) plus Na-adduct peaks that were selectively adsorbed on theIDA-Cu(II) surface. FIG. 5, profile C, and FIG. 6, profile C, show themass spectrum of the same angiotensin I adsorbed on IDA-Cu(II) afterwater wash.

[0049]FIG. 6, profile D, shows the sequential in situ copper-binding (1and 2 Cu) by affinity adsorbed angiotensin I. FIG. 6, profile E, showsthe sequential in situ trypsin digestion of the affinity adsorbedangiotensin I.

[0050]FIG. 7 shows the mass spectrum of myoglobin (4 to 8 fmole)affinity adsorbed on IDA-Cu(II) surface.

[0051]FIG. 8 (top profile) shows the mass spectrum of synthetic caseinpeptide (1934 Da) with multiple phosphorylated forms affinity adsorbedfrom a crude mixture on TED-Fe(III) surface. After sequential in situalkaline phosphatase digestion, only the original nonphosphorylated formremained (lower profile).

[0052]FIG. 9, profile A, shows the mass spectrum of total proteins ininfant formula. FIG. 9, profile B, shows the mass spectrum ofphosphopeptides in infant formula affinity adsorbed on TED-Fe(III)surface. FIG. 9, profile C, shows the mass spectrum of total proteins ingastric aspirate of preterm infant obtained after feeding the infantformula. FIG. 9, profile D, shows the mass spectrum of phosphopeptidesin the gastric aspirate affinity adsorbed on TED-Fe(III) surface.

[0053]FIG. 10A shows the composite mass spectra of human and bovinehistidine-rich glycoprotein adsorbed on IDA-Cu(II) surface before andafter N-glycanase digestion. The mass shifts represent the removal ofcarbohydrate from the respective glycoproteins. FIG. 10B shows thecomposite mass spectra of trypsin digested peptides from thedeglycosylated proteins of the two species (top profile for humanprotein, second from bottom profile for bovine protein) and in situCu(II)-binding of the trypsin digested peptides of the two species(second from top profile for human protein, bottom profile for bovineprotein; the numbers 1, 2 indicate the number of copper bound). FIG. 10Cshows that one such Cu(II)-binding peptide (bottom profile) has at least4 His residues which are specifically modified by diethylpyrocarbonateto form 4 N-carbethoxy-histidyl adducts (1-4, top profile). FIG. 10Dshows the partial C-terminal sequence of the major Cu-binding peptide inthe bovine histidine rich glycoprotein.

[0054]FIG. 11 (bottom profile) shows the mass spectrum of rabbitanti-human lactoferrin immunoglobulin alone (control) affinity adsorbedon sheep anti-rabbit IgG paramagnetic surface. The top profile shows themass spectrum of human lactoferrin and rabbit anti-human lactoferrinimmunoglobulin complex affinity adsorbed on sheep anti-rabbit IgGparamagnetic surface.

[0055]FIG. 12 shows the mass spectrum of human lactoferrin affinityadsorbed from preterm infant urine on a anti-human lactoferrinimmunoglobulin nylon surface. FIG. 13 shows the equivalent mass spectrumof whole preterm infant urine containing 1 nmole/ml of lactoferrin.

[0056]FIG. 14 (lower profile) shows the mass spectrum of pure bovinehistidine rich glycoprotein. The upper profile shows the mass spectrumof bovine histidine rich glycoprotein and fragments affinity adsorbedfrom bovine colostrum on anti-bovine histidine rich glycoproteinimmunoglobulin surface.

[0057]FIG. 15 shows the composite mass spectra of the peptides offollicle stimulating hormone recognized by the different anti-folliclestimulating hormone antibodies.

[0058]FIG. 16 shows the mass spectrum of human lactoferrin affinityadsorbed on a single bead of single-stranded DNA agarose deposited on a0.5 mm diameter probe element.

[0059]FIG. 17 shows the mass spectrum of human lactoferrin affinityadsorbed from preterm infant urine on single-stranded DNA surface

[0060]FIG. 18A shows the composite mass spectra of the total proteins inhuman duodenal aspirate (lower profile) and the trypsin affinityadsorbed from the aspirate on a soybean trypsin inhibitor surface (upperprofile). FIG. 18B shows the mass spectrum of trypsin affinity adsorbedfrom 1 ul of aspirate on a soybean trypsin inhibitor nylon surface.

[0061]FIG. 19A shows the mass spectrum of biotinylated insulin affinityadsorbed from human urine on a Streptavidin surface. FIG. 19B shows themass spectrum of biotinylated insulin affinity adsorbed from humanplasma on a Streptavidin surface.

[0062]FIG. 20 (upper profile) shows the mass spectrum of total proteinsin human serum. FIG. 20 (lower profile) shows the mass spectrum of serumalbumin affinity adsorbed from human serum on a Cibacron-blue surface.

[0063] SEND

[0064]FIG. 21 shows the molecular structure of surface boundcinnamamide; R represents the surface plus cross-linker.

[0065]FIG. 22 (upper profile) shows the mass spectrum of peptidemixtures desorbed from surface bound cinnamamide. FIG. 20B (lowerprofile) shows the mass spectrum of the same peptide mixtures with freecinnamamide.

[0066]FIG. 23 shows the molecular structure of surface bound cinnamylbromide; R represents the surface plus cross-linker.

[0067]FIG. 24 (upper profile) shows the mass spectrum of peptidemixtures desorbed from surface bound cinnamyl bromide. FIG. 22B (lowerprofile) shows the mass spectrum of the same peptide mixtures with freecinnamyl bromide.

[0068]FIG. 25 shows the molecular structure of surface boundMAP-dihydroxybenzoic acid; R represents the surface plus cross-linker.

[0069]FIG. 26 (upper profile) shows the mass spectrum of peptidemixtures desorbed from surface bound MAP alone. FIG. 26 (lower profile)shows the mass spectrum of the same peptide mixtures desorbed fromsurface bound MAP-dihydroxybenzoic acid.

[0070]FIG. 27A shows the mass spectrum (1,200-50,000 m/z region) ofmyoglobin desorbed from surface bound α-cyano-4-hydroxycinnamic acid.FIG. 25B shows the same mass spectrum in the low mass region (0-1200(m/z).

[0071]FIG. 28 shows the molecular structure of energy absorbingmolecules bound to polyacrylamide or nylon or acrylic surface viaglutaraldehyde activation.

[0072]FIG. 29 shows the molecular structure of energy absorbingmolecules bound to polyacrylamide or nylon or acrylic surface viadivinyl sulfone activation.

[0073]FIG. 30 shows the molecular structure of energy absorbingmolecules bound to polyacrylamide or nylon or acrylic surface viadicyclohexylcarbodiimide activation.

[0074]FIG. 31 shows the molecular structure of energy absorbingmolecules bound to polyacrylamide or nylon or acrylic surface withmultiple antigenic peptide via dicyclohexylcarbodiimide activation.

[0075]FIG. 32 shows the molecular structure of thiosalicylic acid boundto iminodiacetate (IDA)-Cu(II) surface.

[0076]FIG. 33 shows the mass spectrum of human estrogen receptordimerization domain desorbed from thiosalicylic acid-IDA-Cu(II) surface.

[0077]FIG. 34 shows the molecular structure of α-cyano-4-hydroxycinnamicacid bound to DEAE surface.

[0078]FIG. 35 shows the mass spectrum of human estrogen receptordimerization domain desorbed from sinapinic acid-DEAE surface. FIG. 33Bshows the mass spectrum of myoglobin desorbed fromα-cyano-4-hydroxycinnamic acid DEAE surface.

[0079]FIG. 36 shows the molecular structure of α-cyano-4-hydroxycinnamicacid bound to polystyrene surface.

[0080] SEPAR

[0081]FIG. 37 shows the C-terminal sequence analysis of surfaceimmobilized via photolytic bond histidine rich glycoprotein metalbinding domain.

DETAILED DESCRIPTION OF THE INVENTION

[0082] It will be apparent to one skilled in the art that varioussubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and the spirit of the invention.

[0083] The development of new MS probe element compositions withsurfaces that allow the probe element to actively participate in thecapture and docking of specific analytes has recently defined severalnew opportunities in the area now being described as Affinity MassSpectrometry (AMS). In brief, several types of new MS probe elementshave been designed (Hutchens and Yip, Rapid Commun Mass Spectrom, 7:576-580 (1993)) with Surfaces Enhanced for Affinity Capture (SEAC). Todate, SEAC probe elements have been used successfully to retrieve andtether different classes of biopolymers, particularly proteins, byexploiting what is known about protein surface structures andbiospecific molecular recognition.

[0084] Progress in structural biology continues to be limited by theinability to obtain biopolymer sequence information at an acceptablerate or level of sensitivity. By utilizing the methods and apparatus ofthe present invention, it has been demonstrated that AMS provides anopportunity to relieve this limitation. Because the immobilized affinitycapture devices on the MS probe element surface (i.e., SEAC) determinesthe location and affinity (specificity) of the analyte for the probesurface, the subsequent analytical AMS process is much more efficientfor several reasons. First, the location of analyte on the probe elementsurface is predetermined. Thus, the subsequent desorption is no longerdependent on a random search of the probe surface matrix field with theincident laser beam. Second, analyte detection sensitivity (and dynamicrange) is increased because molecular ionization suppression effectsoften observed with complex mixtures are eliminated. Third, the tetheredanalyte that is not actually consumed by the initial laser-induceddesorption process remains available for subsequent analyses. Ifexogenous matrix was used to promote analyte desorption, it is removed,in most cases, without loss of the tethered analyte. The remaininganalyte can then be chemically and/or enzymatically modified directly insitu (i.e., while still on the probe element). When analyzed again by MSto determine differences in mass, specific structural details arerevealed. The entire process of analysis/modification can be repeatedmany times to derive structural information while consuming only verysmall quantities of analyte (sometimes only a few femtomoles or less).The demonstrations of protein structure analysis based on AMS have todate included both N— and C-terminal sequence analyses and verificationof several types of sequence-specific posttranslational modificationsincluding phosphorylation and dephosphorylation, glycosylation, cysteineresidue reactivity, site-specific chemical modifications (e.g.,Histidine residues), and ligand binding.

[0085] Beyond biopolymer sequence determinations and the solution ofindividual biopolymer structures, is the ability to understand thestructural determinants of functional supramolecular assemblies. Theopportunity to investigate the structural determinants of higher order(e.g., quaternary) structures is also presented by AMS. It has beendemonstrated by using the present invention that noncovalent molecularrecognition events, some not readily observed by more traditionalbioanalytical procedures (often requiring disruption of equilibrium andstructure dissociating conditions), are investigated directly by theevaluation of molecular associations (i.e., recognition) withmacromolecular analytes that have been tethered, directly or indirectly,to the probe element surface.

[0086] As used herein, “analyte” refers to any atom and/or molecule;including their complexes and fragment ions. In the case of biologicalmacromolecules, including but not limited to: protein, peptides, DNA,RNA, carbohydrates, steroids, and lipids. Note that most importantbiomolecules under investigation for their involvement in the structureor regulation of life processes are quite large (typically severalthousand times larger than H₂O).

[0087] As used herein, the term “molecular ions” refers to molecules inthe charged or ionized state, typically by the addition or loss of oneor more protons (H⁺).

[0088] As used herein, the term “molecular fragmentation” or “fragmentions” refers to breakdown products of analyte molecules caused, forexample, during laser-induced desorption (especially in the absence ofadded matrix).

[0089] As used herein, the term “solid phase” refers to the condition ofbeing in the solid state, for example, on the probe element surface.

[0090] As used herein, “gas” or “vapor phase” refers to molecules in thegaseous state (i.e., in vacuo for mass spectrometry).

[0091] As used herein, the term “analyte desorption/ionization” refersto the transition of analytes from the solid phase to the gas phase asions. Note that the successful desorption/ionization of large, intactmolecular ions by laser desorption is relatively recent (circa 1988)—thebig breakthrough was the chance discovery of an appropriate matrix(nicotinic acid).

[0092] As used herein, the term “gas phase molecular ions” refers tothose ions that enter into the gas phase. Note that large molecular massions such as proteins (typical mass=60,000 to 70,000 times the mass of asingle proton) are typically not volatile (i.e., they do not normallyenter into the gas or vapor phase). However, in the procedure of thepresent invention, large molecular mass ions such as proteins do enterthe gas or vapor phase.

[0093] As used herein in the case of MALDI, the term “matrix” refers toany one of several small, acidic, light absorbing chemicals (e.g.,nicotinic or sinapinic acid) that is mixed in solution with the analytein such a manner so that, upon drying on the probe element, thecrystalline matrix-embedded analyte molecules are successfully desorbed(by laser irradiation) and ionized from the solid phase (crystals) intothe gaseous or vapor phase and accelerated as intact molecular ions. Forthe MALDI process to be successful, analyte is mixed with a freshlyprepared solution of the chemical matrix (e.g., 10,000:1 matrix:analyte)and placed on the inert probe element surface to air dry just before themass spectrometric analysis. The large fold molar excess of matrix,present at concentrations near saturation, facilitates crystal formationand entrapment of analyte.

[0094] As used herein, “energy absorbing molecules (EAM)” refers to anyone of several small, light absorbing chemicals that, when presented onthe surface of a probe element (as in the case of SEND), facilitate theneat desorption of molecules from the solid phase (i.e., surface) intothe gaseous or vapor phase for subsequent acceleration as intactmolecular ions. The term EAM is preferred, especially in reference toSEND. Note that analyte desorption by the SEND process is defined as asurface-dependent process (i.e., neat analyte is placed on a surfacecomposed of bound EAM). In contrast, MALDI is presently thought tofacilitate analyte desorption by a volcanic eruption-type process that“throws” the entire surface into the gas phase. Furthermore, note thatsome EAM when used as free chemicals to embed analyte molecules asdescribed for the MALDI process will not work (i.e., they do not promotemolecular desorption, thus they are not suitable matrix molecules).

[0095] As used herein, “probe element” or “sample presenting device”refers to an element having the following properties: it is inert (forexample, typically stainless steel) and active (probe elements withsurfaces enhanced to contain EAM and/or molecular capture devices).

[0096] As used herein, “MALDI” refers to Matrix-Assisted LaserDesorption/Ionization

[0097] As used herein, “TOF” stands for Time-of-Flight.

[0098] As used herein, “MS” refers to Mass Spectrometry.

[0099] As used herein “MALDI-TOF MS” refers to Matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry.

[0100] As used herein, “ESI” is an abbreviation for Electrosprayionization.

[0101] As used herein, “chemical bonds” is used simply as an attempt todistinguish a rational, deliberate, and knowledgeable manipulation ofknown classes of chemical interactions from the poorly defined kind ofgeneral adherence observed when one chemical substance (e.g., matrix) isplaced on another substance (e.g., an inert probe element surface).Types of defined chemical bonds include electrostatic or ionic (±) bonds(e.g., between a positively and negatively charged groups on a proteinsurface), covalent bonds (very strong or “permanent” bonds resultingfrom true electron sharing), coordinate covalent bonds (e.g., betweenelectron donor groups in proteins and transition metal ions such ascopper or iron), and hydrophobic interactions (such as between twononcharged groups).

[0102] As used herein, “electron donor groups” refers to the case ofbiochemistry, where atoms in biomolecules (e.g, N, S, O) “donate” orshare electrons with electron poor groups (e.g., Cu ions and othertransition metal ions).

[0103] The present invention uses a general category of probe elements(i.e., sample presenting means) with Surfaces Enhanced for LaserDesorption/Ionization (SELDI), within which there are three (3) separatesubcategories. Surfaces Enhanced for Neat Desorption (SEND) where theprobe element surfaces (i.e., sample presenting means) are designed tocontain Energy Absorbing Molecules (EAM) instead of “matrix” tofacilitate desorption/ionizations of analytes added directly (neat) tothe surface. Note that this category 1 (SEND) is used alone or incombination with Surfaces Enhanced for Affinity Capture (SEAC)(category2), where the probe element surfaces (i.e., sample presenting means) aredesigned to contain chemically defined and/or biologically definedaffinity capture devices to facilitate either the specific ornonspecific attachment or adsorption (so-called docking or tethering) ofanalytes to the probe surface, by a variety of mechanisms (mostlynoncovalent). Note that category 2 (SEAC) is used with added matrix orit is used in combination with category 1 (SEND) without added matrix.Thus, the combination of SEND and SEAC actually represents a distinctivecategory.

[0104] Category 3 involves Surfaces Enhanced for Photolabile Attachmentand Release (SEPAR) where the probe element surfaces (i.e., samplepresenting means) are designed/modified to contain one or more types ofchemically defined crosslinking molecules to serve as covalent dockingdevices. These Photolabile Attachment Molecules (PAM) are bivalent ormultivalent in character, that is, one side is first reacted so as topermanently attach the PAM to the probe element surface of the samplepresenting means, then the other reactive side(s) of the PAM is ready tobe reacted with the analyte when the analyte makes contact with thePAM-derivatized probe surface. Such surfaces (i.e., sample presentingmeans) allow for very strong (i.e., stable, covalent) analyte attachmentor adsorption (i.e., docking or tethering) processes that are covalentbut reversible upon irradiation (i.e., photolabile). Such surfacesrepresent platforms for the laser-dependent desorption of analytes thatare to be chemically and/or enzymatically modified in situ (i.e.,directly on the probe tip) for the purpose of structure elucidation.Only those analytes on the probe surface that are actually irradiated(small percentage of total) is desorbed. The remainder of the tetheredanalytes remain covalently bound and is modified without loss due tosome inadvertent uncoupling from the surface. Note that the SEPARcategory (category 3) is characterized by analyte attachment processesthat are reversible upon exposure to light. However, the light-dependantreversal of the analyte surface attachment bond(s) does not necessarilyenable analyte desorption into the gas phase per se. In other words, themolecules responsible for the photolabile attachment of the analytes tothe probe surface are not necessarily the same as the Energy AbsorbingMolecules (EAM) described for SEND. But here is an important exception:The present invention includes some hybrid EAM/PAM chemicals that havedual functionality with respect to SEND and SEPAR. That is, some EAMmolecules presently used for SEND can be modified to act as mediators ofboth the SEND and SEPAR processes. Similarly, some hybrid affinitycapture/PAM chemicals that have dual functionality with respect to SEACand SEPAR are provided. The present invention uses some affinity capturedevices, particularly those that are biologically defined, that aremodified to act as mediators of both the SEAC and SEPAR processes.

[0105] The invention herein presents, a sample presenting means (i.e.,probe element surface) with surface-associated (or surface-bound)molecules to promote the attachment (tethering or anchoring) andsubsequent detachment of tethered analyte molecules in a light-dependentmanner, wherein the said surface molecule(s) are selected from the groupconsisting of photoactive (photolabile) molecules that participate inthe binding (docking, tethering, or crosslinking) of the analytemolecules to the sample presenting means (by covalent attachmentmechanisms or otherwise). Further, a sample presenting means (composedof one or more of the suitable probe element materials described inprevious claims), wherein analyte(s) are bound to the surface saidsample presenting means by one or more photolabile bonds so thatincident pulse(s) of light (e.g., from one or more lasers) is used tobreak the photolabile bond(s) tethering the analyte(s) to the probeelement surface in a manner that is consistent with the subsequentdesorption of the analyte from the stationary (solid) phase surface ofthe probe into the gas (vapor) phase is also presented.

[0106] The chemical specificity(ies) determining the type and number ofsaid photolabile molecule attachment points between the SEPAR samplepresenting means (i.e., probe element surface) and the analyte (e.g.,protein) may involve any one or more of a number of different residuesor chemical structures in the analyte (e.g., His, Lys, Arg, Tyr, Phe,and Cys residues in the case of proteins and peptides). In other words,in the case of proteins and peptides, the SEPAR sample presenting meansmay include probe surfaces modified with several different types ofphotolabile attachment molecules to secure the analyte(s) with aplurality of different types of attachment points.

[0107] The wavelength of light or light intensity (or incident angle)required to break the photolabile attachment(s) between the analyte andthe probe element surface may be the same or different from thewavelength of light or light intensity (or incident angle) required topromote the desorption of the analyte from the stationary phase into thegas or vapor phase.

[0108] The photolabile attachment of the analyte(s) to the probe elementsurface (i.e., sample presenting means), particularly biopolymers suchas peptides, proteins, ribonucleic acid (RNA), deoxyribonucleic acids(DNA), and carbohydrates (CHO), may involve multiple points ofattachment between the probe surface and the analyte macromolecule. Oncethe biopolymer is attached via multiple points of attachment, differentpoints in the backbone of the biopolymer may be deliberately cut orfragmented by chemical and/or enzymatic means so that many of theresulting fragments are now separate and distinct analytes, each onestill attached (tethered) to the probe surface by one or morephotolabile bonds, to be desorbed into the gas phase in parallel forsimultaneous mass analyses with a time-of-flight mass analyzer. Thisprocess enables biopolymer (protein, peptides, RNA, DNA, carbohydrate)sequence determinations to be made.

[0109] As used herein “affinity” refers to physical and/or chemicalattraction between two molecules. Typically used in nature for purposesof structure or regulation of bioactivity (i.e., information transfer).Usually the affinity of one biomolecule for another is quite specific.Used in the present case to describe principle by which molecularanalytes of interest are captured. In the case of SEAC, chemicals orbiomolecules with a characteristic affinity for the analyte(s) ofinterest are tethered (bound) to the surface of the probe element toactively “seek” out and selectively bind the desired analyte.

[0110] As used herein, “molecular recognition” refers to the interactionevent between two molecules with a natural affinity for one another.

[0111] As used herein, “molecular capture” refers to the use of tetheredbiomolecules to attract and bind (capture) other biomolecules for whicha specific affinity relationship exists.

[0112] As used herein, “passive adsorption” refers to the act of simplyplacing the analyte (e.g., with matrix).

[0113] As used herein, “active docking” refers to the deliberate captureof analyte molecules on the surface of an active probe element as in thecase of SEAC.

[0114] As referred to herein “stationary phase” means the same as solidphase. In the present context either the probe element surface itself-orone of the “external” particulate SEND or SEAC devices used inconjunction with an inert probe element surface.

[0115] As used herein, “active surface area” refers to that area of thesurface thought or known to participate in the desired reaction or event(e.g., EAM attachment or affinity capture). The active surface area maybe significantly less than the total surface area (due to physicaleffects such as steric hinderance, some of the total area may not beavailable or useful).

[0116] As used herein, “ligand” refers to a typically relatively smallmolecule (bait) that binds to a large biomolecule (fish). In the presentcase, ligands are attached (chemically bound) through a linker arm(fishing line) to the probe element surface. This process allows thebiomolecular capture event to be localized on the surface (stationary orsolid phase).

[0117] As used herein, “affinity reagent” refers to an analyte capturedevice, viz., the class of molecules (both man made, unnatural, naturaland biological) and/or compounds which have the ability of beingretained on the presenting surface (by covalent bonding, chemicalabsorption, etc.) while retaining the ability of recognition and bondingto an analyte.

[0118] As used herein, “desorption” refers to the departure of analytefrom the surface and/or the entry of the analyte into a gaseous phase.

[0119] As used herein, “ionization” refers to the process of creating orretaining on an analyte an electrical charge equal to plus or minus oneor more electron units.

[0120] As used herein, “adduct” refers to the appearance of anadditional mass associated with the analyte and usually caused by thereaction of excess matrix (or matrix break-down products) directly withthe analyte.

[0121] As used herein, “adsorption”—the chemical bonding (covalentand/or noncovalent) of the energy-absorbing molecules, the affinityreagent (i.e., analyte capture device), and/or the analyte to the probe(presenting surface).

[0122] One embodiment of the present invention is an apparatus formeasuring the mass of an analyte molecule of an analyte sample by meansof mass spectrometry, said apparatus comprising: a spectrometer tube;vacuum means for applying a vacuum to the interior of said tube;electrical potential means within the tube for applying an acceleratingelectrical potential to desorbed analyte molecules from said analytesample;

[0123] sample presenting means removably insertable into saidspectrometer tube, for presenting said analyte sample in associationwith surface associated molecule for promoting desorption and ionizationof said analyte molecules, wherein said surface molecule is selectedfrom the group consisting of energy absorbing molecule, affinity capturedevice, photolabile attachment molecule and combination thereof; ananalyte sample deposited on said sample presenting means in associationwith said surface associated molecules; whereby at least a portion ofsaid analyte molecules not consumed in said mass spectrometry analysiswill remain accessible for subsequent chemical, biological or physicalanalytical procedures; laser beam means for producing a laser beamdirected to said analyte sample for imparting sufficient energy todesorb and ionize a portion of said analyte molecules from said analytesample; and detector means associated with said spectrometer tube fordetecting the impact of accelerated ionized analyte molecules thereon.

[0124] Another embodiment of the present invention is a method in massspectrometry to measure the mass of an analyte molecule, said methodcomprising the steps of: derivitizing a sample presenting surface on aprobe tip face with an affinity capture device having means for bindingwith an analyte molecule; exposing said derivitized probe tip face to asource of said analyte molecule so as to bind said analyte moleculethereto; placing the derivitized probe tip with said analyte moleculesbound thereto into one end of a time-of-flight mass spectrometer andapplying a vacuum and an electric field to form an acceleratingpotential within the spectrometer; striking at least a portion of theanalyte molecules bound to said derivitized probe tip face within thespectrometer with one or more laser pulses in order to desorb ions ofsaid analyte molecules from said tip; detecting the mass of the ions bytheir time of flight within said mass spectrometer; and displaying suchdetected mass. In an preferred embodiment, this method further comprisesapplying a desorption/ionization assisting matrix material to said probetip face in association with said affinity capture device. In a morepreferred embodiment, the method according further comprises removingsaid probe tip from said mass spectrometer; performing a chemical orbiological procedure on said portion of said analyte molecules notdesorbed to alter the composition of said portion of said analytemolecules not desorbed; reinserting said probe tip with said alteredanalyte molecules thereon; and performing subsequent mass spectrometryanalysis to determine the molecular weight of said altered analytemolecules.

[0125] In an additional embodiment, said affinity capture device ischemically bonded to said face of said probe tip, physically adhered tosaid face of said probe tip, adapted to chemically bond to said analytemolecules, or adapted to biologically adhere to said analyte molecules.In a further embodiment, said analyte molecules are biomolecules andsaid affinity reagent is adapted to selectively isolate saidbiomolecules from an undifferentiated biological sample. In a preferredembodiment, said matrix materials are in the weakly acidic to stronglybasic pH range. In a more preferred embodiment, said matrix materialshave a pH above 6.0. Further, an additional embodiment presents the faceof said probe tip formed of an electrically insulating material.

[0126] An additional embodiment of the present invention is a method ofmeasuring the mass of analyte molecules by means of laserdesorption/ionization, time-of-flight mass spectrometry in which anenergy absorbing material is used in conjunction with said analytemolecules for facilitating desorption and ionization of the analytemolecules, wherein the improvement comprises presenting the analytemolecules on or above the surface of the energy absorbing material,wherein at least a portion of the analyte molecules not desorbed in saidmass spectrometry analysis remain chemically accessible for subsequentanalytical procedures.

[0127] A further embodiment of the present invention is an apparatus forfacilitating desorption and ionization of analyte molecules, saidapparatus comprising a sample presenting surface; and surface associatedmolecules, wherein said surface associated molecules are selected fromthe group consisting of energy absorbing molecule, affinity capturedevice, photolabile attachment molecule and combination thereof, saidsurface associated molecules associated with said sample presentingsurface and having means for binding with said analyte molecules.

[0128] In a preferred embodiment, said sample presenting surfacecomprises the surface of a probe tip for use in a time-of-flight massspectrometry analyzer. In addition, the preferred embodiment presents anaffinity capture device or photolabile attachment molecule that ischemically bonded to said sample presenting surface, physically adheredto said sample presenting surface, chemically bonded to said analytemolecules, or is adapted to biologically adhere to said analytemolecules. Further, the preferred embodiment presents analyte moleculesare biomolecules and said affinity capture device or photolabileattachment molecule is adapted to selectively isolate said biomoleculesfrom an undifferentiated biological sample.

[0129] In addition, the apparatus may have a matrix material depositedon said sample presenting surface in association with said affinitycapture device or photolabile attachment molecule. In a more preferredembodiment, the matrix material is in the weakly acidic to stronglybasic pH range. In a most preferred embodiment, the matrix material hasa pH above 6.0. Additionally, a preferred embodiment includes a samplepresenting surface formed of an electrically insulating material.

[0130] In an additional embodiment of the present invention, there ispresented a method for capturing analyte molecules on a samplepresenting surface and desorbing/ionizing said captured analytemolecules from said sample presenting surface for subsequent analysis,said method comprising: derivitizing said sample presenting surface withan affinity capture device or photolabile attachment molecule havingmeans for binding with said analyte molecules; exposing said derivitizedsample present surface to a sample containing said analyte molecules;capturing said analyte molecules on said derivitized sample presentingsurface by means of said affinity capture device or photolabileattachment molecule; and exposing said analyte molecules, while bound tosaid derivitized sample presenting surface by means of said affinitycapture device or photolabile attachment molecule, to an energy or lightsource to desorb at least a portion of said analyte molecules from saidsurface.

[0131] A further embodiment of the present invention is a method forpreparing a surface for presenting analyte molecules for analysis, saidmethod comprising: providing a substrate on said surface for supportingsaid analyte; derivitizing said substrate with an affinity capturedevice or photolabile attachment molecule having means for selectivelybonding with said analyte; and a means for detecting said analytemolecules bonded with said affinity capture device or photolabileattachment molecule. In a preferred embodiment, there is provided theadditional step of applying a detection material to said surface. In amore preferred embodiment, such detection material comprises afluorescing species, an enzymatic species, a radioactive species, or alight-emitting species.

[0132] In an additional preferred embodiment, the step of depositing adesorption/ionization assisting material to said sample presentingsurface in association with said affinity capture device or photolabileattachment molecule is included. In a further preferred embodiment, theenergy source comprises a laser. In another preferred embodiment, anaffinity capture device is used and said energy source comprises an ionsource. Further, a preferred embodiment may include a portion of saidanalyte molecules remaining bound to said sample presenting surfaceafter exposure to said energy source. In a more preferred embodiment,the additional steps of converting at least a portion of the analytemolecules remaining bound on said derivitized sample presenting surfaceto modified analyte molecules by a chemical, biological or physicalreaction, wherein said analyte molecules remain bound to saidderivitized sample presenting surface by means of said affinity capturedevice or photolabile attachment molecule; and exposing said modifiedanalyte molecules to an energy source so as to desorb at least a portionof said modified analyte molecules from said surface are included.

[0133] In an embodiment of the present invention, a sample probe forpromoting desorption of intact analytes into the gas phase comprising: asample presenting surface; and an energy absorbing molecule associatedwith said sample presenting surface, wherein said sample probe promotesdesorption of an intact analyte molecule positioned on, above or betweenthe energy absorbing molecules when said sample probe is impinged by anenergy source is provided. In a more preferred embodiment, the energyabsorbing molecule is selected from the group consisting of cinnamamide,cinnamyl bromide, 2, 5-dihydroxybenzoic acid andα-cyano-4-hydroxycinnamic acid. Also in a preferred embodiment, one mayutilize a sample presenting surface selected from the group consistingof glass, ceramics, teflon coated magnetic materials; organic polymersand native biopolymers.

[0134] In another embodiment of the present invention, there is provideda sample probe for promoting desorption of intact analytes into the gasphase comprising: a sample presenting surface; and an affinity capturedevice associated with said sample presenting surface; wherein, whensaid sample probe is impinged by an energy source, said sample probepromotes the transition of an intact analyte molecule into the gasphase. In a preferred embodiment, the affinity capture device isselected from the group consisting of metal ions, proteins, peptides,immunoglobulins, nucleic acids, carbohydrates, lectins, dyes, reducingagents and combination thereof. In another preferred embodiment, thesample presenting surface is selected from the group consisting ofglass, ceramics, teflon coated magnetic materials; organic polymers andnative biopolymers.

[0135] An additional embodiment presents a sample probe for desorptionof intact analyte into the gas phase, comprising: a sample presentationsurface; and a surface associated molecule wherein said surfaceassociated molecule is a photolabile attachment molecule having at leasttwo binding sites, wherein at least one site is bound to the samplepresentation surface and at least one site is available to bind ananalyte and wherein the analyte binding site is photolabile.

[0136] In another embodiment, there is provided a sample probe forpromoting desorption of intact analytes into the gas phase comprising: asample presentation surface; and either a mixture of at least twodifferent molecules selected from the group consisting of an affinitycapture device, an energy absorbing molecule and a photolabileattachment molecule associated with said sample presentation surface;wherein when an analyte is associated with said sample probe, saidsample probe promotes the transition of the analyte into the gas phasewhen said sample probe is impinged by an energy source; or at least twodifferent affinity capture devices associated with said samplepresentation surface; wherein, when said sample probe is impinged by anenergy source, said sample probe promotes the transition of an analytemolecule into the gas phase at different rates depending on the affinitycapture device associated with said analyte molecule.

[0137] In a preferred embodiment, the analyte is selectively desorbedfrom the mixture after impingement by the energy source. In anotherpreferred embodiment, the affinity devices are arranged in predeterminedarrays. In a more preferred embodiment, the arrays selectively absorb aplurality of different analytes.

[0138] In a more preferred embodiment, an apparatus of the presentinvention is used to quantitate an analyte, wherein the position andquantity of affinity capture devices determines the quantity of analyteabsorbed. In another preferred embodiment, the binding may be selectiveor non-selective.

[0139] In an additional embodiment, a sample probe for promotingdesorption of intact analyte into the gas phase, comprising: a samplepresentation surface; and either a surface associated molecule, whereinsaid surface associated molecule can function both as an energyabsorbing molecule and as an affinity capture device; or a surfaceassociated molecule wherein said surface associated molecule is aphotolabile attachment molecule having at least two binding sites,wherein at least one site is bound to the sample presentation surfaceand at least one site is available to bind an analyte and wherein theanalyte binding site is photolabile.

[0140] A different embodiment of the present invention includes a methodin mass spectrometry to measure the mass of an analyte molecule, saidmethod comprising the steps of: derivitizing a sample presenting surfaceon a probe tip face with a photolabile attachment molecule (PAM),wherein said PAM has at least two binding sites, one binding site bindsto the sample presenting surface and at least one binding site isavailable for binding with an analyte molecule; exposing saidderivitized probe tip face to a source of said analyte molecule so as tobind said analyte molecule thereto; placing the derivitized probe tipwith said analyte molecules bound thereto into one end of atime-of-flight mass spectrometer and applying a vacuum and an electricfield to form an accelerating potential within the spectrometer;striking at least a portion of the analyte molecules bound to saidderivitized probe tip face within the spectrometer with one or morelaser pulses in order to desorb ions of said analyte molecules from saidtip; detecting the mass of the ions by their time of flight within saidmass spectrometer; and displaying such detected mass. In a preferredembodiment, the step of applying a desorption/ionization assistingmatrix material to said probe tip face in association with said PAM isincluded. In a more preferred embodiment, an additional steps ofremoving said probe tip from said mass spectrometer; performing achemical, biological or physical procedure on said portion of saidanalyte molecules not desorbed to alter the composition of said portionof said analyte molecules not desorbed; reinserting said probe tip withsaid altered analyte molecules thereon; and

[0141] performing subsequent mass spectrometry analysis to determine themolecular weight of said altered analyte molecules are included. Apreferred embodiment may also include PAM being chemically bonded tosaid face of said probe tip; PAM being chemically bonded to said analytemolecule, wherein said bond between the PAM and the analyte molecule isbroken and the analyte molecule is released in a light dependent manner;or, where said analyte molecules are biomolecules, said PAM is adaptedto selectively isolate said biomolecules from an undifferentiatedbiological sample. In another preferred embodiment, said matrixmaterials are in the weakly acidic to strongly basic pH range. In a morepreferred embodiment, said matrix materials have a pH above 6.0. Apreferred embodiment may also include the face of said probe tip beingformed of an electrically insulating material.

[0142] A further embodiment presents a method of measuring the mass ofanalyte molecules by means of laser desorption/ionization,time-of-flight mass spectrometry in which a photolabile attachmentmolecule (PAM) is used in conjunction with said analyte molecules forfacilitating desorption and ionization of the analyte molecules, theimprovement comprising: presenting the analyte molecules on or above thesurface of the PAM, wherein at least a portion of the analyte moleculesnot desorbed in said mass spectrometry analysis remain chemicallyaccessible for subsequent analytical procedures.

[0143] Another embodiment of the present invention is a sample probe forpromoting of differential desorption of intact analyte into the gasphase, comprising: a sample presentation surface; and at least twodifferent photolabile attachment molecules associated with said samplepresentation surface; wherein, when said sample probe is impinged by anenergy source, said sample probe promotes the transition of an analytemolecule into the gas phase at different rates depending on thephotolabile attachment molecule associated with said analyte molecule.In a preferred embodiment, the photolabile attachment molecules arearranged in predetermined arrays. In a more preferred embodiment, thearrays selectively absorb a plurality of different analytes.

[0144] An additional embodiment of the present invention includes asample probe for promoting desorption of intact analytes into the gasphase comprising: a sample presenting surface; and a photolabileattachment molecule associated with said sample presenting surface;wherein, when said sample probe is impinged by an energy source, saidsample probe promotes the transition of an intact analyte molecule intothe gas phase. In a preferred embodiment, and analyte is quantitated,wherein the position and quantity of photolabile attachment moleculedetermines the quantity of analyte absorbed.

[0145] Another embodiment shows a method for biopolymer sequencedetermination comprising the steps of: binding a biopolymer analyte toprobe tip containing a sample presenting surface having a surfaceselected molecule selected from the group consisting of an energyabsorbing molecule, an affinity capture device, a photolabile attachmentmolecule and a combination thereof; desorption of biopolymer analyte inmass spectrometry analysis, wherein at least a portion of saidbiopolymer is not desorbed from the probe tip; analyzing the results ofthe desorption modifying the biopolymer analyte still bound to the probetip; and repeating the desorption, analyzing and modifying steps untilthe biopolymer is sequenced. A preferred embodiment presents thebiopolymer selected from the group consisting of protein, RNA, DNA andcarbohydrate.

[0146] The following specific examples describe specific embodiments ofthe present invention and its materials and methods, are illustrative ofthe invention and are not intended to limit the scope of the invention.

[0147] The examples of the present invention utilize a time-of-flightmass spectrometer with a high energy source, such as a laser beam, tovaporize the analyte from the surface of a probe tip. In the process,some of the molecules are ionized. The positively charged molecules arethen accelerated through a short high voltage field and enter into afield-free flight tube. A sensitive detector positioned at the end ofthe flight tube gives a signal as each molecular ion strikes it. Oneskilled in the art recognizes that other modes of detection andionization can also be used.

EXAMPLE 1 Energy Absorbing Molecules in Aqueous, Neutralized Form

[0148] Prior art matrix material used in matrix-assisted laserdesorption time-of-flight mass spectrometry are strongly acidic. One ofthe present discoveries is that analytes is desorbed when mixed withneutralized energy absorbing molecules dissolved in entirely aqueoussolvents. By suitable neutralization to pH 6.0 or above, the matrixmaterial is made largely passive to subsequent chemical or enzymaticreactions carried out on the analyte molecules presented on the probetip surfaces. Since only a small fraction of the analyte molecules areused in each desorption/mass spectrometer measurement, the samples onthe probe tips are available for in situ sequential chemical orenzymatic modifications. After modification the samples are analyzed bymass spectrometry. Analysis on the same probe tips provides a moreaccurate determination of the molecule and its characteristics,including its structure.

[0149] Mass spectrometry is performed on a modified Vestec model VT2000or a MAS model SELDI Research Linear time-of-flight mass spectrometerwhich uses a frequency-tripled output from a Q-switched neodymiumyttriumaluminum garnet (Nd-YAG) pulsed laser (355 nm, 5 ns pulse). Ionsdesorbed by pulsed laser irradiation are accelerated to an energy of 30keV and allowed to drift along a 2-meter field free drift region(maintained at 10⁻⁸ torr). Ion signals detected using a 20-stagediscrete dynode electron multiplier are amplified by a factor of 10using a fast preamplifier prior to being recorded using a 200 MS/stransient recorder (LeCroy TR8828D, 8-bit y-axis resolution) or aTektronix digitizer capable of fast signal averaging. The lasterirradiance is adjusted real-time, while monitoring the process on anoscilloscope (Tektronix), in order to achieve optimum ion signal. Datareduction (peak centroid calculations and time to mass/chargeconversions) are performed with PC-based software. A VG TOFSpec massspectrometer which uses a nitrogen laser generating pulsed laser at 335nm. or a Linear LDI 1700 mass spectrometer which uses a nitrogen lasergenerating pulsed laser 335 nm. may also be used.

[0150] I. Specific Analysis

[0151] 1. Sinapinic acid (Aldrich Chemical Co., Inc., Milwaukee, Wis.)is suspended in water at 20 mg/ml (pH 3.88) and neutralized withtriethylamine (Pierce, Rockford, Ill.) to pH 6.2-6.5. An aqueous mixture(1 μl) of synthetic peptides, containing human histidine richglycoprotein metal-binding domains (GHHPH)₂G(1206 Da), (GHHPH)₅G (2904Da), and human estrogen receptor dimerization domain (D473-L525) (6168.4Da) is mixed with 2 μl sinapinic acid (20 mg/ml water, pH 6.2) on aprobe tip and analyzed by laser desorption time-of-flight massspectrometry. After acquiring five spectra (average 100 laser shots perspectrum), the probe is retrieved, 2 μl of 20 mM Cu(SO)₄ is added andthe sample is reanalyzed by mass spectrometry. FIG. 1A (upper profile)shows the mass spectrum of the three peptides desorbed in the presenceof neutralized energy absorbing molecules. FIG. 1B (lower profile) showsthe in situ metal-binding of the peptides in the presence of neutralenergy absorbing molecules. The (GHHPH)₂G peptide can bind at least 4Cu(II), the (GHHPH)₅G peptide can bind at least 5 Cu(II) and thedimerization domain can bind at least 1 Cu(II) under the presentexperimental conditions. Similar result is obtained withα-cyano-4-hydroxycinnamic acid (20 mg/ml water) neutralized to pH 6.5.

[0152] 2. An aliquot of 1 μl of human β casein phosphopeptide(R1−K18+5P) (2488 Da) is mixed with 1 μl of sinapinic acid (20 mg/mlwater) neutralized to pH 6.5, and analyzed by laser desorptiontime-of-flight mass spectrometry. After acquiring five spectra (average100 laser shots per spectrum), the probe is removed, the remainingphosphopeptide mixed with the neutralized sinapinic acid is digesteddirectly on the probe tip by 0.5 μl of alkaline phosphatase (Sigma) andincubated at 23° C. for 5 min. After acquiring five spectra (average 100laser shots per spectrum), the probe is removed, further digestion onremaining phosphopeptides is carried out by adding another aliquot of0.5 μl of alkaline phosphatase and incubated at 23° C. for 5 min. Thesample is re-analyzed by laser desorption mass spectrometry. FIG. 2A(top profile) shows the mass spectrum of the phosphopeptide desorbed inthe presence of neutralized energy absorbing molecules. FIG. 2B (secondfrom top profile) shows the in situ 5 min alkaline phosphatase digestionto remove phosphate groups from the phosphopeptide. The 0P, 1P and 3Ppeaks represent the products after removal of five, four and twophosphate groups respectively from the phosphopeptide. FIG. 2C (thirdfrom top profile) shows that further in situ digestion with alkalinephosphatase can result in almost complete removal of all phosphategroups from the phosphopeptide. In contrast, FIG. 2D (bottom profile)shows that in the control experiment where in situ alkaline phosphatase(0.5 μl) digestion is carried out in the presence of energy absorbingmolecules without prior neutralization (e.g. sinapinic acid at pH 3.88or dihydroxybenzoic acid at pH 2.07), very limited digestion occurred in10 min at 23° C.

[0153] 3. An aliquot of 1 μl of (GHHPH)₅G peptide (2904 Da) is mixedwith 2 μl of sinapinic acid (20 mg/ml water) neutralized to pH 6.2, andanalyzed by laser desorption time-of-flight mass spectrometry. Afteracquiring five spectra (average 100 laser shots per spectrum), theremaining peptides mixed with neutralized sinapinic acid are digesteddirectly on the probe tip by 1 μl of carboxypeptidase P (BoehringerMannheim Corp, Indianapolis, Ind.) and incubated at 23° C. for 30 min.The sample is analyzed by mass spectrometry. FIG. 3 shows a compositemass spectra of the peptide before (lower profile) and after (upperprofile) in situ digestion by carboxypeptidase P in the presence ofneutralized energy absorbing molecules. The decrease in mass representsthe removal of a Gly residue from the C-terminal of the peptide.

[0154] These examples illustrate that neutralized energy absorbingmolecules in aqueous solutions are more biocompatible in preserving thestructure and function of the analytes even when added in large molarexcess. Their presence results in no interference to in situ sequentialchemical or enzymatic reactions on the remaining analyte.

EXAMPLE 2 Nonmetallic Probe Elements (Sample Presenting Surfaces)

[0155] It has been found that the probe elements (probe tips or samplepresenting surfaces) used in the process of the invention need not bemetal or metal-coated, as described in prior art procedures. The samplepresenting surfaces are composed of a variety of materials, includingporous or nonporous materials, with the porous materials providingsponge-like, polymeric, high surface areas for optimized adsorption andpresentation of analyte.

[0156] Polypropylene or polystyrene or polyethylene or polycarbonate aremelted in an open flame and deposited as a thin layer on a 2 mm diameterstainless steel probe element so as to cover it completely. Solid glassrod or solid nylon filaments (up to 1.5 mm diameter) or polyacrylamiderod are cut into 1 cm segments and inserted into the stainless steelprobe support. Magnetic stir bars (1.5×8 mm, teflon-coated) are insertedinto stainless steel probe tip support. An aliquot of 1 μl of peptidemixture containing (GHHPH)₅G and human estrogen receptor dimerizationdomain, is mixed with 2 μl of dihydroxybenzoic acid (dissolved in 30%methanol, 0.1% trifluoroacetic acid) on each of such probe elements andanalyzed by laser desorption time-of-flight mass spectrometry. FIG. 4shows that analytes could be desorbed from several examples ofinsulating, biocompatible surfaces.

[0157] These surfaces can be derivatized (at varying densities) to bindby chemical bonds (covalent or noncovalent) affinity adsorption reagents(affinity capture devices), energy absorbing molecules (bound “matrix”molecules) or photolabile attachment molecules. The geometry of thesample presenting surface is varied (i.e., size, texture, flexibility,thickness, etc.) to suit the need (e.g., insertion into a livingorganism through spaces of predetermined sizes) of the experiment(assay).

EXAMPLE 3 Affinity-Directed Laser Desorption (Surface Enhanced AffinityCapture, SEAC)

[0158] This example describes the use of existing and new solid phaseaffinity reagents designed for the (1) capture (adsorption) of one ormore analytes, (2) the preparation of these captured analytes (e.g.,washing with water or other buffered or nonbuffered solutions to removecontaminants such as salts, and multiple cycles of washing, such as withpolar organic solvent, detergent-dissolving solvent, dilute acid, dilutebase or urea), and (3) most importantly, the direct transfer of thesecaptured and prepared analytes to the probe surface for subsequentanalyte desorption (for detection, quantification and/or mass analysis).Affinity capture devices are immobilized on a variety of materials,including electrically insulating materials (porous and nonporous),flexible or nonrigid materials, optically transparent materials (e.g.,glass, including glass of varying densities, thicknesses, colors andwith varying refractive indices), as well as less reactive, morebiocompatible materials (e.g., biopolymers such as agarose, dextran,cellulose, starches, peptides, and fragments of proteins and of nucleicacids). The preferred probe tip, or sample surface, for selectiveadsorption/presentation of sample for mass analysis are (1) stainlesssteel (or other metal) with a synthetic polymer coating (e.g.,cross-linked dextran or agarose, nylon, polyethylene, polystyrene)suitable for covalent attachment of specific biomolecules or othernonbiological affinity reagents, (2) glass or ceramic, and/or (3)plastics (synthetic polymer). The chemical structures involved in theselective immobilization of affinity reagents to these probe surfaceswill encompass the known variety of oxygen-dependent, carbon-dependent,sulfur-dependent, and/or nitrogen-dependent means of covalent ornoncovalent immobilization.

[0159] I. Surface Immobilized Metal Ion as the Affinity Capture Device

[0160] 1. Cu(II) ion is chelated by iminodiacetate (IDA) groupcovalently attached to either porous agarose beads (Chelating SepharoseFast Flow, Pharmacia Biotech Inc., Piscataway, N.J., ligand density22-30 μmole/ml gel) or solid silica gel beads (Chelating TSK-SW,ToyoSoda, Japan, ligand density 15-20 μmole/ml gel). A mixture ofsynthetic peptides containing neurotensin (1655 Da), sperm activatingpeptide (933 Da) and angiotensin I (1296.5 Da), is mixed with 50 μlpacked volume of TSK-SW IDA-Cu(II) at pH 7.0 (20 mM sodium phosphate,0.5 M sodium chloride) at 23° C. for 10 min. The gel is separated fromthe remaining peptide solution by centrifugation and is then washed with200 μl sodium phosphate, sodium chloride buffer, pH 7.0 three times toremove nonspecifically bound peptides. Finally, the gel is suspended in50 μl of water. Aliquots of 2 μl gel suspension and nonadsorbed peptidesolution are mixed with 1 μl of sinapinic acid (dissolved in methanol)on a stainless steel probe tip and analyzed by laser desorptiontime-of-flight mass spectrometry. After acquiring five spectra (averageof 100 laser shots per spectrum) on various spots of the probe tip, thesinapinic acid is removed by methanol. An aliquot of 2 μl of 20 mM CuSO₄is added, then mixed with 1 μl of sinapinic acid and reanalyzed by laserdesorption time-of-flight mass spectrometry. After acquiring anotherfive spectra (average of 100 laser shots per spectrum) on various spotsof the probe tip, the sinapinic acid is removed by methanol. Theremaining peptide adsorbed on IDA-Cu(II) gel beads is then digested with1 μl of trypsin (Sigma) in 0.1 M sodium bicarbonate, pH 8.0 at 23° C.for 10 min in a moist chamber. The gel beads are then washed with waterto remove enzyme and salt before 1 μl of sinapinic acid is added and thesample analyzed by laser desorption time-of-flight mass spectrometry.FIG. 5A shows the molecular ions (and multiple Na-adducts) of spermactivating factor (933 Da) and neurotensin (1655 Da) in the remainingpeptide solution unabsorbed by the IDA-Cu(II). There is no significantpeak corresponding to angiotensin I (1296.5 Da). The mass spectrum inFIG. 5B shows the angiotensin I plus Na-adduct peaks that areselectively adsorbed on the IDA-Cu(II) gel. When the IDA-Cu(II) gel isfurther washed with 500 μl of water two times, the resulting massspectrum shows only the parent angiotensin I ion and no other adductpeaks (FIGS. 5 and 6, profiles C). FIG. 6D shows the in situ copperbinding (1 and 2 Cu) by the angiotensin peptide. FIG. 6E shows the insitu trypsin digestion of the angiotensin peptide at the single Arg2position in the sequence.

[0161] This example illustrates that: a) laser desorption issuccessfully carried out on analyte affinity adsorbed onsurface-immobilized metal ion; b) once bound, the surface is washed withvarious solvents to remove all contaminating compounds in the sample togive a very clean mass spectrum of the analyte; c) the affinity capturedevice selects only the analyte of defined structure (in this caseangiotensin I is selectively adsorbed from the peptide mixture byIDA-Cu(II) because this peptide has a free N-terminal and two histidineamino acid residues in the sequence, both properties are required forstrong Cu(II)-binding, whereas both sperm activating factor andneurotensin have blocked N-terminal and no histidine amino acid residuesin their sequences); d) structure and function analyses throughsequential in situ chemical or enzymatic modifications is carried out onthe adsorbed analyte with minimal loss at each step of reaction andwash; and e) a probe element with surface bound substrate (e.g.,angiotensin I) is used to monitor specific enzyme activity (e.g.,trypsin) in situ (e.g., inside the gastrointestinal tract of the humanbody).

[0162] 2. A solution of horse heart myoglobin (325 pmole, 16,952 Da) ismixed with 50 μl of TSK-SW IDA-Cu(II) at pH 7.0 (20 mM sodium phosphate,0.5 M sodium chloride) at 23° C. for 10 min. The gel is separated fromthe solution by centrifugation and then washed with 500 μl of buffer twotimes and 500 μl of water two times. The quantity of remaining myoglobinin all these solutions are then estimated spectrophotometrically, thequantity adsorbed on the gel can then be calculated. The gel issuspended in 50 μl of water and then serially diluted into water. Analiquot of 0.5 μl of the diluted gel suspension is mixed with 1 μl ofsinapinic acid (dissolved in 30% methanol, 0.1% trifluoroacetic acid)and analyzed by laser desorption time-of-flight mass spectrometry. FIG.7 shows that a detectable signal (signal/noise=6, after averaging 50laser shots) of myoglobin is obtained with a calculated quantity of 4 to8 fmole deposited on the probe tip.

[0163] This example illustrates that affinity adsorbed analytes on asurface are much more easier to transfer and are free from any loss bynonspecific adsorption to container and transfer device surfaces. Theadsorbed analyte is sequestered on predetermined areas (that are evenless than the laser spot size) of the sample presenting surface in low(atto to femtomole) quantities at a defined surface density or localconcentration required for the efficient detection by laserdesorption/ionization time-of-flight mass spectrometry.

[0164] 3. The human β casein peptides (E2-K18) are synthesized on anApplied Biosystem Model 430A Peptide Synthesizer using the NMP-HOBtprotocol. The Ser residues to be phosphorylated are coupled to thepeptide chain without side chain protecting group. The unprotected Serare first phosphinylated usingdi-t-butyl-N,N,-diisopropyl-phosphoramidite. The phosphite ester is thenoxidized with t-butyl peroxide, washed, and cleaved from the resin. Allthe side chain protecting groups are removed with 95% trifluoroaceticacid. The crude phosphopeptides are extracted with methyl t-butyl etherand dried. This crude preparation of synthetic phosphopeptides isdissolved in 50 mM MES, 0.15 M sodium chloride, pH 6.5 and mixed with 50μl of tris(carboxymethyl)-ethylenediamine (TED)-Fe(III) immobilized onporous Sepharose (synthesized as described by Yip, T.-T. and Hutchens,T. W., Protein Expression and Purification 2: 355-362 (1991), liganddensity 65 μmole/ml) at 23° C. for 15 min. The gel is washed with 500 μlof the same buffer three times and then with 500 μl of water once. Analiquot of 1 μl of gel is mixed with 1 μl of sinapinic acid (dissolvedin 30% methanol, 0.1% trifluoroacetic acid) on the probe tip andanalyzed by laser desorption time-of-flight mass spectrometry. Afteracquiring five spectra (average of 100 laser shots per spectrum) onvarious spots of the probe tip, the sinapinic acid is removed bymethanol, and the remaining phosphopeptides adsorbed on TED-Fe(III) isdigested directly on the probe tip by 1 μl of alkaline phosphatase(ammonium sulfate suspension, Sigma) in 50 mM HEPES pH 7.0 at 23° C. for10 min. in a moist chamber. The gel is washed with water to removeenzyme and salt. Sinapinic acid is added and the sample is reanalyzed bylaser desorption time-of-flight mass spectrometry. FIG. 8 (top profile)shows the distribution of casein peptide (1934 Da) with multiplephosphorylated forms. After in situ alkaline phosphatase digestion, onlythe original nonphosphorylated form remains (lower profile).

[0165] This example illustrates the application of SEAC as a quickmonitor of phosphopeptide synthesis in a crude mixture without priorcleanup. The identity of the phosphopeptide is readily confirmed by insitu alkaline phosphatase digestion.

[0166] 4. Aliquots of 100 μl of preterm infant formula (SIMILAC, MeadeJohnson) and gastric content of preterm infant aspirated 90 min afterfeeding of the formula are mixed with 50 μl of TED-Fe(III) Sepharose in0.1 M MES, 0.15 M sodium chloride, pH 6.5 at 23° C. for 15 min. The gelis washed with 500 μl of the same buffer three times and then with 500μl of water once. Aliquots of 1 μl of gel suspensions or preterm infantformula or gastric aspirate are mixed with 2 μl of sinapinic acid(dissolved in 50% acetonitrile, 0.1% trifluoroacetic acid) on the probetip and analyzed by laser desorption time-of-flight mass spectrometry.FIG. 9 shows that the mass spectrum of whole gastric aspirate (secondfrom top profile) is quite similar to that of whole infant formula(bottom profile) in the 1,000-15,000 Da region. However, the massspectra of analytes selectively adsorbed by TED-Fe(III) from the twosamples are quite different, there are more low molecular weightphosphopeptides (i.e., bound by TED-Fe(III)) present in the gastricaspirate (top profile) than in the formula (second from bottom profile)due to the gastric proteolytic digestion of phosphoproteins present inthe formula.

[0167] This example illustrates that SEAC is particularly useful inanalyzing specific analytes in biological samples. Phosphopeptides aremore difficult to detect in the presence of other contaminatingcomponents in a complex sample because they are less ionized in thepositive ion mode. However, when the phosphopeptides are selectivelyadsorbed and all other components in the sample are removed, no suchsignal depression occurs.

[0168] 5. Aliquots of 200 μl of human and bovine histidine-richglycoprotein are mixed with 50 μl of IDA-Cu(II) Sepharose (Pharmacia) atpH 7.0 (20 mM sodium phosphate, 0.5 M sodium chloride) at 23° C. for 10min. The gel is washed with 500 μl buffer two times and 500 μl wateronce. Aliquots of 1 μl of gel are mixed with 2 μl of sinapinic acid(dissolved in 30% methanol 0.1% trifluoroacetic acid) and analyzed bylaser desorption time-of-flight mass spectrometry. After acquiring fivespectra (average of 100 laser shots per spectrum) on various spots ofthe probe tip, the sinapinic acid is removed by methanol wash. Theremaining glycoproteins adsorbed on the IDA-Cu(II) gel is then digestedwith N-glycanase in 20 mM sodium phosphate, 0.5 M sodium chloride, 3 Murea, pH 7.0 at 37° C. overnight in a moist chamber. After washing withwater to remove enzyme and salt, 2 μl of sinapinic acid is added and thesample is analyzed by mass spectrometry. After acquiring five spectra(average of 100 laser shots per spectrum) on various spots of the probetip, the sinapinic acid is removed by methanol. Aliquots of 2 μl oftrypsin in 0.1 M sodium bicarbonate are added and incubated at 37° C.for 30 min in a moist chamber. After a water wash to remove enzyme andsalt, sinapinic acid is added and the sample is analyzed by massspectrometry. After acquiring five spectra (average of 100 laser shotsper spectrum) on various spots of the probe tip, the sinapinic acid isremoved by methanol. Aliquots of 2 μl of 20 mM CuSO₄ is added. This isfollowed by addition of 2 μl of sinapinic acid and then analyses by massspectrometry. After acquiring five spectra (average of 100 laser shotsper spectrum) on various spots of the probe tip, the sinapinic acid isremoved by methanol. Aliquots of 2 μl of diethylpyrocarbonate (Sigma) in5 mM HEPES, pH 6.5 are added and incubated at 23° C. for 30 min. After awater wash to remove chemicals and buffer salts, 2 μl of sinapinic acidis added and the sample is analyzed by mass spectrometry. To obtain apartial sequence of the metal-binding peptides, instead of modifying thehistidine residues with diethylpyrocarbonate, add 1 ul ofcarboxypeptidase Y (Boehringer Mannheim) to the tryptic digest adsorbedon the surface and incubate at room temperature in a moist chamber for 5min. Wash away the enzyme and salt with water, add 1 ul of sinapinicacid and analyze by mass spectrometry. FIG. 10A shows the composite massspectra of human and bovine histidine-rich glycoprotein adsorbed onIDA-Cu(II) Sepharose before and after N-glycanase digestion. The massshifts represent the removal of carbohydrate from the respectiveglycoproteins. FIG. 10B shows the composite mass spectra of trypsindigested peptides from the deglycosylated proteins of the two species(top profile for human protein, second from bottom profile for bovineprotein) and in situ Cu(II)-binding of the trypsin digested peptides ofthe two species (second from top profile for human protein, bottomprofile for bovine protein; the numbers 1, 2 indicate the number ofcopper bound). FIG. 10C shows that one such Cu(II)-binding peptide(bottom profile) has at least 4His residues which are specificallymodified by diethylpyrocarbonate to form 4 N-carbethoxy-histidyl adducts(1-4, top profile). FIG. 10D shows the partial C-terminal sequence ofthe major Cu-binding peptide in the bovine histidine rich glycoprotein.This example illustrates the effective use of SEAC to probe thestructure and function of metal-binding domains of proteins fromdifferent species.

[0169] II. Surface Immobilized Antibody as the Affinity Capture Device

[0170] 1. Polyclonal rabbit anti-human lactoferrin antibody is customgenerated against purified human lactoferrin by Bethyl Laboratories(Montgomery, Tex.). The antibody is affinity-purified by thiophilicadsorption and immobilized lactoferrin columns. Sheep anti-rabbit IgGcovalently attached to magnetic beads are obtained from Dynal AS, Oslo,Norway (uniform 2.8 μm supermagnetic polystyrene beads, ligand density10 μg sheep IgG per mg bead). Human lactoferrin (1 nmole, ⁶⁹Fe-labeled,81,100 Da) is incubated with rabbit anti-human lactoferrin antibody in20 mM sodium phosphate, 0.15 M sodium chloride, pH 7.0 at 37° C. for 30min. Subsequently, 40 μl of sheep anti-rabbit IgG on Dynabeads (6-7×10⁸beads/ml) is added and incubated at 37° C. for 30 min. The beads arewashed with 500 μl of sodium phosphate buffer three times and 500 μlwater two times. The final amount of human lactoferrin bound to thecomplex is estimated to be 4 pmole. Approximately one-tenth of the beadsis transferred to a teflon-coated magnetic probe tip, mixed with 2 μl ofsinapinic acid (dissolved in 30% methanol, 0.1% trifluoroacetic acid)and analyzed by laser desorption time-of-flight mass spectrometry. FIG.11 shows the presence of lactoferrin (81,143 Da) in the antigen-primaryantibody-secondary antibody complex (upper profile), whereas the primaryantibody-secondary antibody control (lower profile) shows only therabbit antibody signal (149,000 Da for singly charged, and 74,500 Da forthe doubly charged).

[0171] This example illustrates that a) laser desorption is successfullycarried out on analyte affinity-adsorbed on surface immobilized antibody(if the analyte signal is unambiguously identified in a mixture ofprimary antibody-analyte complex, any capture device, e.g., surfaceimmobilized secondary antibody, Protein A, Protein G, Streptavidin, ofthe primary antibodies is used in this method of identifying theanalyte); b) the principle of protein discovery via specific molecularrecognition events where one of the analytes is detected through itsassociation with the primary target of capture; and c) the use ofmagnetic surface as efficient capture device.

[0172] 2. Affinity-purified rabbit anti-human lactoferrin is covalentlybound to the tip of an activated nylon probe element (2 mm diameter) viaglutaraldehyde. This is immersed in 1 ml of preterm infant urine, pH7.0, containing 350 fmole of human lactoferrin and stirred at 4-8° C.for 15 hr. The nylon probe tip is removed and washed with 1 ml of 20 mMsodium phosphate, 0.5 M sodium chloride, 3 M urea, pH 7.0 three timesand 1 ml of water two times. An aliquot of 2 μl of sinapinic acid(dissolved in 30% methanol, 0.1% trifluoroacetic acid) is added and thesample is analyzed by laser desorption time-of-flight mass spectrometry.FIG. 12 shows the human lactoferrin molecular ion (signal/noise=2.5,average of 25 laser shots) in the mass spectrum. FIG. 13 shows theequivalent mass spectrum of whole preterm infant urine containing 1nmole/ml of lactoferrin; the signal suppression caused by the presenceof other components in the urine sample is so severe that even additionof several thousand fold excess over 350 fmole/ml of lactoferrin asdescribed for FIG. 12 can not be detected.

[0173] This example illustrates the use of a SEAC device on a flatsurface (a two-dimensional configuration) of a flexible probe element.This SEAC device may be used to isolate target analyte materials fromundifferentiated biological samples such as blood, tears, urine, saliva,gastrointestinal fluids, spinal fluid, amniotic fluid, bone marrow,bacteria, viruses, cells in culture, biopsy tissue, plant tissue orfluids, insect tissue or fluids, etc. The specific affinity adsorptionstep cleaned up the analyte from contamination by other components in acomplex sample and thus overcome the signal depression effect especiallywhen the analyte is present in very low concentration (femtomole/ml).

[0174] 3. Further improvement of detection sensitivity by the SEACtechnique is achieved by amplification of a label bound to the analyte.One way of doing this is by the combination of enzyme catalysis and thestreptavidin-biotin system. After capturing minute quantities oflactoferrin on a nylon probe element as described in Example 3.II.2.biotinylated anti-lactoferrin antibody or biotinylated single-strandedDNA is used to bind specifically to the lactoferrin. Streptavidin isthen added to bind specifically to the biotinylated label. Finallybiotinylated alkaline phosphatase is added to bind specifically to thestreptavidin. Since several such biotinylated alkaline phosphatase canbind to one streptavidin, there is a primary level of amplification. Thesecond level of amplification comes from the enzyme catalysis where theenzyme can achieve a turnover number of 10² to 10³ min⁻¹. Assay ofalkaline phosphatase enzyme activity can easily be accomplished by usinga low molecular weight phosphorylated substrate such as ATP, NADPH or aphosphopeptide. The efficiency of detecting the mass shift of a lowmolecular weight analyte is much higher than that of a 80 kDaglycoprotein.

[0175] 4. The ultimate improvement of detection at the present moment isachieved by the amplification based on the polymerase chain reactionprinciple. After capturing minute quantities of lactoferrin on a nylonprobe element as described in Example 3.II.2. biotinylatedanti-lactoferrin antibody or biotinylated single-stranded DNA is used tobind specifically to the lactoferrin. Streptavidin is then added to bindspecifically to the biotinylated label. A piece of biotinylated linearDNA is finally added to bind to the streptavidin. This bound DNA labelis amplified in a 30-cycle polymerase chain reaction procedure. Eachcycle consists of a 1 min denaturation step at 94° C., a 1 min annealingreaction at 58° C., and a 1 min primer extension reaction at 72° C. Thistechnique provides amplification factors in the 10⁶ fold range. Theamplified DNA is detected directly by laser desorption mass spectrometryusing 3—OH picolinic acid as the matrix.

[0176] 5. Polyclonal rabbit anti-bovine histidine rich glycoproteinantibody is custom generated against purified bovine histidine richglycoprotein by Bethyl Laboratories (Montgomery, Tex.). The antibody isaffinity-purified by thiophilic adsorption and immobilized bovinehistidine rich glycoprotein columns. The purified antibody isimmobilized on AffiGel 10 (BioRad Laboratories, Hercules, Calif., liganddensity 15 μmole/ml gel) according to manufacturer's instruction. Analiquot of 200 μl of bovine colostrum is diluted with 200 μl of 20 mMsodium phosphate, pH 7.0 and mixed with 50 μl of immobilized antibody at23° C. for 30 min. The gel is washed with 500 μl of 20 mM sodiumphosphate, 0.5 M sodium chloride, 3 M urea, pH 7.0 three times and 500μl of water two times. An aliquot of 1 μl of the washed gel is mixedwith 2 μl of sinapinic acid (dissolved in 30% methanol, 0.1%trifluoroacetic acid) on the probe tip and analyzed by laser desorptiontime-of-flight mass spectrometry. FIG. 14 shows the composite massspectra of purified bovine histidine rich glycoprotein (lower profile)and proteins affinity adsorbed from bovine colostrum (upper profile).The result indicates the presence of intact histidine rich glycoproteinand its major proteolytic fragments in bovine colostrum.

[0177] This example illustrates the effective use of SEAC as a fast andsimple technique to detect and characterize new proteins in a smallquantity of biological fluid. This result supports the initial findingsobtained by the very labor-intensive technique of immunoblotting ofpolyacrylamide gel electrophoresis.

[0178] 6. Antibody epitope mapping is easily achieved with the SEACtechnique. Three different sources of anti-human follicle stimulatinghormone (a polyclonal specific against beta FSH from ChemiconInternational, Temecula, Calif., a monoclonal specific against beta 3epitope from Serotec, Indianapolis, Ind., a monoclonal from Biodesign,Kennebunk, Me.) are immobilized on AffiGel 10 according tomanufacturer's instruction. These immobilized antibodies are all testedto bind specifically the follicle stimulating hormone by incubating withtwo different preparations of follicle stimulating hormone (a semipurepreparation from Chemicon, and a crude preparation from AccurateChemical and Scientific Corp.) and then analyzed by mass spectrometry inthe presence of sinapinic acid. Then the semipure preparation of humanFSH (Chemicon) is digested with trypsin and separate aliquots (7 ul) arereacted with the immobilized antibodies (10 ul of 1:1 gel suspension) inphosphate-buffered saline at 4° C. for 2 hr. After washing withphosphate-buffered saline and water, the adsorbed proteins are analyzedby laser desorption mass spectrometry in the presence of sinapinic acid.FIG. 15 shows the composite mass spectra of the peptides of folliclestimulating hormone recognized by the different antibodies. The twomonoclonal antibodies clearly recognize different epitopes, whereas thepolyclonal recognizes multiple epitopes common to those recognized byboth monoclonals.

[0179] III. Surface Immobilized Nucleic Acid as the Affinity CaptureDevice

[0180] 1. Single-stranded DNA immobilized on 4% agarose beads areobtained from GIBCO BRL (Gaithersburg, Md., ligand density 05-1.0 mgDNA/ml gel). An aliquot of ¹²⁵I-human lactoferrin (equivalent to 49nmole) is mixed with 100 μl of immobilized single-stranded DNA in 20 mMHEPES, pH 7.0 at room temperature for 10 min. The gel is washed with 500μl of HEPES buffer five times and then suspended in equal volume ofwater. The amount of lactoferrin bound per bead is estimated to be 62fmole by determining the radioactivity and counting the number of beadsper unit volume. Various numbers of beads (from 1 to 12) are depositedon 0.5 mm diameter probe tips, mixed with 0.2 μl of sinapinic acid(dissolved in 30% methanol, 0.1% trifluoroacetic acid) and analyzed bylaser desorption time-of-flight mass spectrometry. FIG. 16 shows themass spectrum of lactoferrin affinity adsorbed on a single bead ofsingle-stranded DNA agarose. This is a representative spectrum from atotal of five (average of 100 laser shots per spectrum) obtained fromthe single bead.

[0181] This example illustrates that laser desorption is successfullycarried out on analyte affinity adsorbed on surface immobilizedbiopolymer such as nucleic acid. The specificity of interaction betweenhuman lactoferrin and DNA has been documented and effectively exploitedin capturing minute quantities of lactoferrin from preterm infant urine.In this case, the combination of the efficiency of transferring thelactoferrin affinity capture device with the sensitivity of laserdesorption mass spectrometry greatly increases the sensitivity ofdetection.

[0182] 2. An aliquot of 1 ml of preterm infant urine containing 30 pmoleof ⁵⁹Fe-human lactoferrin is mixed with 20 μl of single-stranded DNAagarose in 0.1 M HEPES pH 7.4 at 23° C. for 15 min. The gel is washedwith 500 μl of HEPES buffer two times and 500 μl of water two times. Thegel is suspended in equal volume of water and 1 μl of the suspension(containing not more than 350 fmole of adsorbed lactoferrin asdetermined by radioactivity) is mixed with 1 μl of sinapinic acid(dissolved in 30% methanol, 0.1% trifluoroacetic acid) on a probe tipand analyzed by laser desorption time-of-flight mass spectrometry. FIG.17 shows the mass spectrum of lactoferrin extracted from urine bysurface immobilized DNA as the affinity capture device.

[0183] This example illustrates the efficiency and sensitivity ofdetecting minute quantities of high molecular weight analyte inbiological fluid with the DNA capture device.

[0184] IV. Surface Immobilized Miscellaneous Biomolecule as the AffinityCapture Device

[0185] 1. Soybean trypsin inhibitor (Sigma) is immobilized on AffiGel 10(BioRad) according to manufacturer's instructions. An aliquot of 100 μlof human duodenal aspirate is mixed with 50 μl of surface immobilizedsoybean trypsin inhibitor at pH 7.0 (20 mM sodium phosphate, 0.5 Msodium chloride) at 23° C. for 15 min. The gel is then washed with 500μl of phosphate buffer three times and 500 μl of water two times.Aliquots of 1 μl of gel suspension or the original duodenal aspirate aremixed with 2 μl of sinapinic acid (dissolved in 50% acetonitrile, 0.1%trifluoroacetic acid) and analyzed by laser desorption time-of-flightmass spectrometry. FIG. 18A shows the composite mass spectra of thetotal duodenal aspirate (lower profile) and the proteins adsorbed bysurface immobilized soybean trypsin inhibitor (upper profile). The majorpeak in the affinity captured sample represents trypsin. Similar resultsare obtained with only 1 μl of duodenal fluid deposited on a) the tip ofa nylon probe element coupled to soybean trypsin inhibitor viaglutaraldehyde and b) the tip of an acrylic probe element coated withpolyacrylamide coupled to soybean trypsin inhibitor via eitherglutaraldehyde or divinyl sulfone (FIG. 18B).

[0186] These results indicate a) the unambiguity in detecting andcharacterizing a specific analyte in biological fluids and b) thefeasibility of in situ sampling by inserting a flexible (e.g. nylon)probe element through an endoscope directly into the human body (e.g.small intestine) for diagnostic purposes.

[0187] 2. Streptavidin immobilized on Dynabead (uniform, 2.8 μm,superparamagnetic, polystyrene beads) is obtained Dynal, AS, Oslo,Norway. Aliquots of 150 μl of human plasma or urine containing 18 pmoleof biotinylated insulin (Sigma) are mixed with 20 μl suspension ofstreptavidin Dynabead at pH 7.0 (20 mM sodium phosphate, 0.5 M sodiumchloride) at 23° C. for 10 min. The beads are then washed with 500 μlbuffer containing 3M urea three times and 500 μl water once. Aliquots of0.5 μl of the bead suspension are mixed with 2 μl of sinapinic acid(dissolved in 30% methanol, 0.1% trifluoroacetic acid) and analyzed bylaser desorption time-of-flight mass spectrometry. FIG. 19A shows themass spectrum of biotinylated insulin affinity adsorbed from urine. Themultiple peaks represent insulin derivatized with one to three biotingroups. FIG. 19B shows the mass spectrum of biotinylated insulinaffinity adsorbed from plasma.

[0188] This example illustrates that laser desorption is carried out onanalyte affinity adsorbed via the biotin-streptavidin binding. In viewof the tight binding between biotin and avidin (Ka=10¹⁵ M⁻¹), thissystem serves as an ideal SEAC device for proteins and nucleic acid on aprobe surface where in situ sequential chemical and enzymaticmodifications are performed.

[0189] 3. Human estrogen receptor DNA-binding domain (84 residues) isexpressed in bacteria. The plasmid expression vector pT₇ERDBD (J.Schwabe, MRC Laboratory of Molecular Biology, Cambridge, UK) istransformed into E. coli BL21(DE3)plyS cells (Novagene). Expression ofthe DNA binding domain is induced by 1 mM isopropylthiogalactoside(GIBCO BRL) and the bacteria are harvested after induction for 3 hr.Whole induced bacteria are analyzed directly by matrix-assisted laserdesorption/ionization mass spectrometry to verify that the DNA-bindingdomain is the major peptide synthesized. The peptide is purified byreverse phase HPLC from the bacterial lyzate, and immobilized on AffiGel10 (BioRad). A 30-bp DNA sequence containing the estrogen responseelement is synthesized by Genosys (Houston, Tex.). Interaction ofsurface affinity adsorbed apo-, Zn— and Cu-bound forms of DNA-bindingdomain with sequence specific nucleic acid (estrogen response element)are studied on glass probe elements using 3-hydroxypicolinic acid as thematrix.

[0190] This example illustrates the use of protein surface functionaldomain as capture device in SEAC. The effect of metal-binding on thestructure and function of such protein surface domains can beinvestigated.

[0191] 4. Different aliquots of lectins immobilized on surfaces (e.g.,Con A-Sepharose, wheat germ lectin-Sepharose, Pharmacia) are used tobind the glycopeptides in human and bovine histidine-rich glycoproteintryptic digests. After washing with buffers and water to remove unboundpeptides, sequential enzyme digestion are performed in situ with FUCaseI, MANase I, HEXase I, NANase III and PNGase (Glyko, Inc, Novato,Calif.). The samples are analyzed with laser desorption time-of-flightmass spectrometry to study the carbohydrate composition of theglycopeptides in the two proteins. This example illustrates the use ofSEAC device to tether a glycopeptide, the carbohydrate component ofwhich can then be sequenced in situ.

[0192] V. Surface Immobilized Dye as the Affinity Capture Device

[0193] Cibacron Blue 3GA-agarose (Type 3000, 4% beaded agarose, liganddensity 2-5 μmoles/ml gel) is obtained from Sigma. An aliquot of 200 μlof human plasma is mixed with 50 μl of surface immobilized Cibacron Blueat pH 7.0 (20 mM sodium phosphate, 0.5 M sodum chloride) at 23° C. for10 min. The gel is then washed with 500 μl of buffer three times and 500μl of water two times. An aliquot of 1 μl of gel suspension is mixedwith 2 μl of sinapinic acid (dissolved in 50% acetonitrile, 0.1%trifluoroacetic acid) and analyzed by laser desorption time-of-flightmass spectrometry. FIG. 20 shows the selective adsorption of human serumalbumin (doubly charged ion [M+2H]²⁺, 32,000 m/z, singly charged ion[M+H]⁺, 64,000 m/z, dimer ion, 2[M+H]⁺, 128,000 m/z) from the serumsample by surface immobilized Cibacron Blue (lower profile). Otherimmobilized dyes tested included Reactive Red 120-agarose, ReactiveBlue-agarose, Reactive Green-agarose, Reactive Yellow-agarose (all fromSigma) and each selects different proteins from human plasma.

EXAMPLE 4 Surface Enhanced Neat Desorption (SEND)

[0194] This example describes the method for desorption and ionizationof analytes in which the analyte is not dispersed in a matrixcrystalline structure but is presented within, on or above an attachedsurface of energy absorbing molecules in a position where it isaccessible and amenable to a wide variety of chemical, physical andbiological modification or recognition reactions. The surface isderivatized with the appropriate density of energy absorbing moleculesbonded (covalently or noncovalently) in a variety of geometries suchthat mono layers and multiple layers of attached energy absorbingmolecules is used to facilitate the desorption of analyte molecules ofvarying masses.

[0195] The Examples shown below (Groups I-IV) demonstrate the combinedSEND and SEAC where the adsorbed (bonded) energy absorbing moleculesalso act as affinity adsorption reagents to enhance the capture ofanalyte molecules.

[0196] I. Energy Absorbing Molecules Bound by Covalent Bond to theSurface

[0197] 1. Cinnamamide (Aldrich) (not a matrix at laser wavelength of 355nm by prior art) is dissolved in isopropanol: 0.5 M sodium carbonate(3:1) and mixed with divinyl sulfone (Fluka, Ronkonkoma, N.Y.) activatedSepharose (Pharmacia) at 23° C. for 2 hr. The excess energy absorbingmolecules are washed away with isopropanol. The proposed molecularstructure is presented in FIG. 21. Aliquots of 2 μl of the bound or freemolecules are deposited on the probe tips, 1 μl of human estrogenreceptor dimerization domain in 0.1% trifluoroacetic acid is added ontop and analyzed by laser desorption time-of-flight mass spectrometry.The result shows that peptide ion signals are detected only on the boundenergy absorbing molecule surface (FIG. 20, top profile), the freemolecules are not effective (FIG. 20, bottom profile).

[0198] 2. Cinnamyl bromide (Aldrich) (not a matrix at laser wavelengthof 355 nm by prior art) is dissolved in isopropanol:0.5 M sodiumcarbonate (3:1) and mixed with divinyl sulfone (Fluka) activatedSepharose at 23° C. for 15 hr. The excess energy absorbing molecules arewashed away with isopropanol. The proposed molecular structure ispresented in FIG. 23. Aliquots of 2 μl of the bound or free moleculesare deposited on the probe tips, 1 μl of peptide mixtures in 0.1%trifluoroacetic acid is added on top and analyzed by laser desorptiontime-of-flight mass spectrometry. The result shows that peptide ionsignals are detected only on the bound energy absorbing molecule surface(FIG. 24, top profile), the free molecules are not effective (FIG. 24,bottom profile).

[0199] 3. Dihydroxybenzoic acid is activated by dicyclohexylcarbodiimideand mixed with Fmoc-MAP 8 branch resin (Applied Biosystems, ForsterCity, Calif.) at 23° C. for 15 hr. The excess energy absorbing moleculesare washed away by methanol. The proposed molecular structure ispresented in FIG. 25. Aliquots of 1 μl of the MAP 8 branch surface withand without bound energy absorbing molecules are deposited on the probetips, 1 μl of peptide mixtures in 0.1% trifluoroacetic acid was added ontop and analyzed by laser desorption time-of-flight mass spectrometry.The result shows that peptide ion signals are detected only on thesurface with bound energy absorbing molecules (FIG. 26, bottom profile),the control surface without any energy absorbing molecules is noteffective (FIG. 24, top profile).

[0200] 4. α-cyano-4-hydorxycinnamic acid is dissolved in methanol andmixed with AffiGel 10 or AffiGel 15 (BioRad) at various pHs at 23° C.for 2-24 hours. The excess energy absorbing molecules are washed away bymethanol. Aliquots of 2 μl of the bound molecules are deposited on theprobe tips, 1 μl of peptide mixtures or myoglobin, or trypsin orcarbonic anhydrase is added on top and analyzed by laser desorptiontime-of-flight mass spectrometry. The result shows that myoglobin ionsignal is detected on the surface with bound energy absorbing molecules(FIG. 27A) with very little contaminating low mass ion signals (FIG.27B).

[0201] 5. A 40% polyacrylamide solution is prepared and cast into thedesired shape of a probe tip. The gel is allowed to air dry until nonoticeable reduction in size is observed. The tip is submerged into a 9%glutaraldehyde/buffer (v/v) solution and incubated with gentle shakingat 37° C. for 2 hours. After incubation, buffer is used to rinse offexcess glutaraldehyde. The activated tip is added to a saturatedbuffered energy absorbing molecule solution and incubated at 37° C.(approx.) for 24 hours (approx.) with gentle shaking. Organic solventsare used to solubilize the energy absorbing molecules in situations thatrequired it. The tip is rinsed with buffer and placed into a 9%ethanolamine/water (v/v) solution to incubate at 25° C. with gentleshaking for 30 minutes. Next, the tip is rinsed with buffer and added toa 5 mg/mL solution of sodium cyanoborohydride/buffer to incubate at 25°C. for 30 minutes. Finally, the tip is rinsed well with buffer andstored until use. The same reaction is carried out on nylon tips whichis prepared by hydrolysis with 6N HCl under sonication for 2 minutes andthen rinsed well with water and buffer. The same reaction is alsoperformed on acrylic tips activated by soaking in 20% NaOH for 7 dayswith sonication each day for 30-60 min and then washed. The proposedgeneral molecular structure of the surface is shown in FIG. 28.

[0202] 6. A 40% polyacrylamide solution is prepared and cast into thedesired shape of a probe tip. The gel is air dried until no noticeablereduction in size is observed. A 0.5 M sodium carbonate buffer with a pHof 8.8 is prepared as rinsing buffer. The tip is next placed into asolution of divinyl sulfone (Fluka) and buffer at a ratio of 10:1,respectively and incubated for 24 hours. The tip is rinsed with bufferand placed into an energy absorbing molecule buffered solution at a pHof 8 to incubate for 2 hours. The same reaction is carried out on nylontips which is prepared by hydrolysis with 6N HCl under sonication for 2minutes and then rinsed well with water and buffer. The same reaction isalso performed on acrylic tips activated by soaking in 20% NaOH for 7days with sonication each day for 30-60 min and then washed. Theproposed general molecular structure of the surface is shown in FIG. 29.

[0203] 7. A 40% polyacrylamide solution is prepared and cast into thedesired shape of a probe tip. The gel is air dried until no noticeablereduction in size is observed. An energy absorbing molecule solution at100 mg/mL in dichloromethane/NMP (2:1 respectively) and a 1Mdicyclohexylcarbodiimide/NMP solution are mixed at a ratio of 1:2(EAM:DCC), respectively. The EAM/DCC solution is next incubated at 25°C. for 1 hour while stirring. After incubation, a white precipitate isobserved. The white precipitate is filtered in a sintered glass filter.The flow through is the DCC activated EAM. Next, the tip is placed intothe DCC activated EAM solution and incubated at 25° C. for 2 hours(approx.). The tip is finally rinsed with a variety of solvents such asacetone, dichloromethane, methanol, NMP, and hexane. The same reactionis carried out on nylon tips which is prepared by hydrolysis with 6N HClunder sonication for 2 minutes and then rinsed well with water andbuffer. The same reaction is also performed on acrylic tips activated bysoaking in 20% NaOH for 7 days with sonication each day for 30-60 minand then washed. The proposed general molecular structure of the surfaceis shown in FIG. 30.

[0204] 8. A 40% polyacrylamide solution is prepared and cast into thedesired shape of a probe tip. The gel is air dried until no noticeablereduction in size was observed. A 100 mg/mL solution ofN-α-Fmoc-N-ε-Fmoc-L-lysine in dichloromethane/NMP (2:1 respectively) anda 1M DCC/NMP solution are mixed at a ratio of 1:2 (lysine:DCC),respectively. The lysine/DCC solution is incubated at 25° C. for 1 hourwhile stirring. After incubation, a white precipitate is observed andfiltered with a sintered glass filter. The flow through is DCC activatedlysine. The tip is placed into the DCC activated lysine solution andincubated at 25° C. for 2 hours (approx.). The tip is next placed into 5mL of piperidine and incubated at 25° C. for 45 minutes with gentlestirring. DCC activated lysine is repeatedly reacted in consecutivecycles with the tip until the desired lysine branching is attained. AnEAM solution at 100 mg/mL in dichloromethane/NMP (2:1 respectively) anda 1M DCC/NMP solution are mixed at a ratio of 1:2 (EAM:DCC),respectively. The EAM/DCC solution is incubated at 25° C. for 1 hourwhile stirring. After incubation, a white precipitate is observed andfiltered with a sintered glass filter. The flow through is the DCCactivated EAM. The EAM contains an acid functional group that reactswith the DCC. The tip is placed into the DCC activated EAM solution andincubated at 25° C. for 2 hours (approx.) with gentle shaking. Finally,the tip is rinsed with excess dichloromethane, NMP, and methanol beforeuse. The same reaction is carried out on nylon tips which is prepared byhydrolysis with 6N HCl under sonication for 2 minutes and then rinsedwell with water and buffer. The same reaction is also performed onacrylic tips activated by soaking in 20% NaOH for 7 days with sonicationeach day for 30-60 min and then washed. The proposed general molecularstructure of the surface is shown in FIG. 31.

[0205] II. Energy Absorbing Molecules Bound by Co-Ordinate Covalent Bondto the Surface

[0206] 1. Thiosalicylic acid (Aldrich) is dissolved in either water or50% methanol in water or methanol. These solutions are either used assuch or the pH of the solutions is adjusted to 6.5 with 0.5 M sodiumbicarbonate or ammonium hydroxide or triethylamine. Cu(II) ion arechelated by either iminodiacetate (IDA) (Chelating Sepharose Fast Flow,Pharmacia) or tris(carboxymethyl)ethyleneidamine (TED) (synthesized asdescribed by Yip and Hutchens, 1991) immobilized on gel surface. Thesolutions of energy absorbing molecule are mixed with the IDA-Cu(II) orTED-Cu(II) gel at 40 to 23° C. for 5 min to 15 hours. The excess energyabsorbing molecules are washed away with either water or 50% methanol inwater or methanol. The proposed molecular structure of the surface isshown in FIG. 32. Aliquots of 1 μl of the bound energy absorbingmolecules are deposited on the probe tips, 1 μl of peptide mixtures orestrogen receptor dimerization domain or myoglobin in 0.1%trifluoroacetic acid is added on top and analyzed by laser desorptiontime-of-flight mass spectrometry. FIG. 33 shows one representative massspectrum of estrogen receptor dimerization domain desorbed from thissurface.

[0207] 2. Sequential in situ reactions are readily accomplished onsamples deposited on top of an EAM surface. Thiosalicylic acidco-ordinate covalently bound to IDA-Cu(II) on a probe surface isprepared as described in Section 2.1. An aliquot of 1 μl of (GHHPH)₅Gpeptide is deposited on the surface and analyzed by laser desorptiontime-of-flight mass spectrometry. After obtaining several spectra (eachan average of 50 laser shots), the sample is removed. An aliquot of 2 μlof carboxypeptidase Y (Boehringer Mannheim) is added directly on thesurface and incubated at 37° C. in a moist chamber for 5 min to 1 hr.The in situ enzyme digestion is terminated by 1 μl of 0.1%trifluoroacetic acid and the sample is reanalyzed by mass spectrometry.

[0208] 3. Another illustration of sequential in situ reaction is trypsindigestion followed by C-terminal sequencing. Thiosalicylic acidco-ordinate covalently bound to IDA-Cu(II) on a probe surface isprepared as described in Section 2.1. An aliquot of 1 μl of estrogenreceptor dimerization domain (6168.4 Da) is deposited on the surface andanalyzed by laser desorption time-of-flight mass spectrometry. Afterobtaining several spectra (each an average of 20 laser shots), thesample is removed. An aliquot of 2 μl of trypsin (Sigma) in 0.1M sodiumbicarbonate is added on the surface and incubated at 37° C. for 15 min.The in situ enzyme digestion is terminated by 1 μl of 0.1%trifluoroacetic acid and the sample is reanalyzed by mass spectrometry.After obtaining several spectra (each an average of 20 laser shots), thesample is removed. An aliquot of 2 μl of carboxypeptidase Y (BoehringerMannheim) is added directly on the surface and incubated at 37° C. in amoist chamber for 1 hr. The in situ enzyme digestion is terminated by 1μl of 0.1% trifluoroacetic acid and the sample is reanalyzed by massspectrometry.

[0209] III. Energy Absorbing Molecules Bound by Ionic Bond to theSurface

[0210] Sinnapinic acid or α-cyano-4-hydroxycinnamic acid are suspendedin water and the pH is adjusted to 6.6 with dilute sodium hydroxide.Tentacle DEAE Fractogel (EM Separations, Gibbstown, N.J.) is washed with20 mM HEPES, pH 6.0 and suction dried. The energy absorbing moleculessolution is mixed with the DEAE gel at 23° C. for 15 hours. The gel iswashed with water until all excess energy absorbing molecules wereremoved. The proposed molecular structure of the surface is shown inFIG. 34. An aliquot of 0.5 μl of the bound energy absorbing molecules isdeposited on the probe tips, 1 μl of estrogen receptor dimerizationdomain or myoglobin in 0.1% trifluoroacetic acid is added on top andanalyzed by laser desorption time-of-flight mass spectrometry. FIGS. 35Aand B show the mass spectra.

[0211] IV. Energy Absorbing Molecules Bound by Hydrophobic/Van der WaalsBonds to the Surfaces

[0212] 1. α-cyano-4-hydroxcinnamic acid is dissolved in 50% methanol inwater and dimethylsulfoxide. This is mixed with aminomethylatedpolystyrene at 23° C. for 15 hours. The excess energy absorbingmolecules are washed away with 50% methanol in water. The proposedmolecular structure is shown in FIG. 36. An aliquot of 1 μl of the boundenergy absorbing molecules is deposited on the probe tip, 1 μl ofpeptide is added on top and analyzed by laser desorption time-of-flightmass spectrometry.

EXAMPLE 5 Surfaces Enhanced for Photolabile Attachment and Release(SEPAR)

[0213] The linear assembly of individual building blocks (monomers) thatdefine the structure and characteristics of biopolymers such as DNA,RNA, and protein are often unknown but are decoded or sequenced (inwhole or in part) with a method that involves differential massdeterminations of partially digested (i.e., chemical or enzymatic)biopolymer analytes by laser desorption/ionization time-of-flight (TOF)mass spectrometry (MS).

[0214] Given biopolymers are first coupled to the SELDI probe elementsurface through one or more (multiple) covalent photolytic (i.e., lightsensitive) bonds. Next, various number of individual units (monomers) atthe ends of the biopolymer are cleaved (i.e., removed) in a singlereaction by enzymatic or chemical methods. The analytes remaining on theprobe element surface consist of a variety (population) of mass-definedbiopolymers with different numbers of their end monomer units missing. Asmall but sufficient portion of the modified biopolymers are uncoupled(untethered) from the probe element surface by laser light, that is, bycleavage of the photolytic bonds with UV light between 260 nm and 365nm. The uncoupled biopolymers are desorbed/ionized by time-of-flightmass spectrometry.

[0215] I. Coupling of Biopolymers to the SELDI Surface

[0216] Three components are involved: 1) a surface that is activated toreact with either amine or carboxyl groups, or both; 2) photolyticcompound, typically azo-based compound of the general formula R₁—N═N—R₂,e.g., 5-(4-aminophenylazo)salicylic acid (Aldrich), azodicarbonamide(Aldrich), or other mechanisms generating such photolytic bond such asthe active hydrogen reactive chemistries with diazonium compounds areused; and 3) biopolymer, e.g., proteins, nucleic acids, carbohydrates.

[0217] A photolytic compound must first be attached to activatedsurface, e.g., azodicarbonamide to amine-reactive surfaces,aminophenylazosalicylic acid to either amine or carboxyl reactivesurfaces. Then activate either photolytic compound or biopolymer by oneof many conventional chemistries, e.g., amine reactivechemistries—cyanogen bromide, N-hydroxysuccinimide esters, FMPactivation, EDC-mediated, divinyl sulfone; hydroxyl reactivechemistries—epoxy activation, divinyl sulfone; sulfhydryl reactivechemistries—iodoacetyl activation, maleimide, divinyl sulfone, epoxyactivation; carbonyl reactive chemistries—hydrazide, reductiveamination; active hydrogen reactive chemistries—diazonium, which alsogenerate a photolytic azo bond at the same time. Finally, attach thebiopolymer to photolytic compound through one or more (multiple) bonds.Wash away the excess chemicals with aqueous and organic solvents, highionic strength and low pH solvents in multiple cycles.

[0218] II. Mass Spectrometric Analysis to Verify Structural Integrity

[0219] UV laser from 260 to 365 nm will cleave the photolytic bond. Theuncoupled biopolymers are desorbed/ionized by MALDI TOF (one skilled inthe art knows that SEND, SEAC and SEPAR may also be used).

[0220] III. In situ Sequencing of Biopolymer

[0221] This is accomplished by any of the well-known sequentialdegradation with enzymatic or chemical methods, e.g., N-terminalsequencing of proteins with aminopeptidase, C-terminal sequencing ofproteins with carboxypeptidase, N-terminal sequencing of proteins withEdman degradation; sequencing of nucleic acids with exonuclease,sequencing of nucleic acids with Sanger's method; sequencing ofcarbohydrate with specific enzymes such as neuraminidase, mannase,fucase, galactosidase, glucosidase, O— or N-glycanase. After washing toremove excess reagent and reaction products, the analytes remainingtethered on the surface via multiple photolytic bonds consisting of apopulation of mass-defined biopolymers with different numbers of theirend monomer missing are analyzed by MALDI TOF MS (one skilled in the artknows that SEND, SEAC and SEPAR may also be used).

[0222] Multiple internal sequencing with enzymatic or chemical methods,e.g., cleavage of proteins with endoprotease or cyanogen bromidefollowed by sequential degradation of N— and/or C-terminal; cleavage ofnucleic acids with endonuclease followed by sequential degradation withexonuclease or chemical method; cleavage of polysaccharide chains withendoglycosidase H or endoglycosidase F followed by sequential cleavagewith specific enzymes. After washing to remove excess reagent andreaction products, the analytes remaining on the surface consisting ofmultiple populations of mass-defined biopolymers with different numbersof their end monomer missing are analyzed by MALDI TOF MS (one skilledin the art knows).

[0223] IV. Specific Examples of Sequencing

[0224] A demonstration of this principle is provided by the actual aminoacid sequence determination of a 26-residue peptide:

[0225] GHHPHGHHPHGHHPHGHHPHGHHPHGHHPHG.

[0226] This peptide (GHHPH)₆G defines the metal-binding domain withinthe intact sequence of the 80-kDa protein known as histidine-richglycoprotein (HRG).

[0227] Glass beads with surface arylamine groups as coupling ligands(Sigma) are washed with and suspended in cold 0.3M HCl. A 50 mg/mLaqueous solution of NaNO₂ is added to the beads at a ratio of 1:5 (v/v)(NaNO₂:HCl) and incubated at 4° C. for 15 minutes with gentle shaking.After incubation, the beads are washed with cold 0.3M HCl and 50 mMsodium phosphate buffer pH 8.0. The peptide to be sequenced is added tothe beads in sodium phosphate buffer at pH 8.0 and incubated for 24 hrs.at 4° C. with gentle shaking. The beads with coupled peptides are washedwith sodium phosphate buffer, sodium phosphate buffer with highconcentration of salt (e.g., 1.0 M), dilute acid and organic solvent(e.g., methanol) until no peptide signal is detected in the supernate byMALDI-TOF mass spectrometry (one skilled in the art knows SEND, SEAC,and SEPAR may also be used) or by absorbance at 220 nm. An aliquot of 1μL of the beads is then deposited on the probe tip, 1 μL of sinapinicacid (dissolved in 50% methanol/0.1% trifluoroacetic acid) is mixed withthe beads and the sample was analyzed by laser desorption time-of-flightmass spectrometry. After obtaining several spectra (each an average of50 laser shots), the remaining peptides on the surface are washed freeof sinapinic acid with methanol and then digested with carboxypeptidaseY (Boehringer Mannheim) at 23° C. in a moist chamber. The digestedpeptides are next washed with phosphate buffered saline (PBS) pH 8.0. Analiquot of 1 μL of sinapinic acid is added to the surface and analyzedagain by laser desorption time-of-flight mass spectrometry. The resultof the C-terminal sequence analysis of the GHHPHG sequence is shown inFIG. 35. A nascent sequence of the peptide is observed. The sequence isdeduced by the differences in the mass between two peaks.

[0228] The second example is the simultaneous sequencing of multiplepeptides covalently bound by photolytic bonds to a surface. Humanestrogen receptor dimerization domain (6168.4 Da) is tethered to thesurface via multiple photolytic bonds. The peptide has three methionineresidues in its sequence and are cleaved specifically by cyanogenbromide to generate peptides of masses 2170.58 Da (D1-M18), 3118.77 Da(A19-M45), 535.62 Da (S46-M50) and 397.62 Da (E51-L53). All thesepeptides are bound to the surface via the photolytic bonds. Each ofthese are subsequently digested in situ with carboxypeptidase Y togenerate a nascent sequence that is completely resolved from the other.

[0229] Another approach to protein structure determination issimultaneous N-terminal sequencing of multiple peptides generated bytryptic digest of a protein coupled to a surface by multiple photolyticbonds. Insulin B chain is tethered to the surface via multiplephotolytic bonds. The peptide has two lysine/arginine residues in itssequence that are cleaved specifically by trypsin to generate peptidesof masses 2585.9 Da (F1-R22) and 859.0 Da (G23-K29), both of which arebound to the surface via the photolytic bonds. Each of these aresubsequently subjected in situ to either aminopeptidase digestion ormultiple cycles of Edman degradation to generate a nascent sequence thatis completely resolved from the other.

[0230] Coupling and sequencing of nucleic acids is performed withsimilar procedure. Glass beads with surface arylamine groups as couplingligands (Sigma) are washed with and suspended in cold 0.3M HCl. A 50mg/mL aqueous solution of NaNO₂ is added to the beads at a ratio of 1:5(v/v) (NaNO₂:HCl) and incubated at 4° C. for 15 minutes with gentleshaking. After incubation, the beads are washed with cold 0.3M HCl and50 mM sodium phosphate buffer pH 8.0. The DNA (e.g., estrogen receptorresponsive element, a 30-base pair oligonucleotide) to be sequenced isadded to the beads in sodium phosphate buffer at pH 8.0 and incubatedfor 24 hrs. at 4° C. with gentle shaking. The beads with coupled DNA arewashed with sodium phosphate buffer, sodium phosphate buffer with highconcentration of salt (e.g., 1.0 M), dilute acid and organic solvent(e.g., methanol) until no DNA signal is detected in the supernate byMALDI-TOF mass spectrometry (one skilled in the art knows that SEND,SEAC and SEPAR may also be used) or by absorbance at 260 nm. An aliquotof 1 μL of the beads is then deposited on the probe tip, 1 μL of3-hydroxypicolinic acid (dissolved in 50% methanol/0.1% trifluoroaceticacid) is mixed with the beads and the sample is analyzed by laserdesorption time-of-flight mass spectrometry. After obtaining severalmass spectra (each an average of 50 laser shots), the remaining DNAbound on the surface are washed free of 3-hydroxypicolinic acid withmethanol and digested with exonuclease (Boehringer Mannheim) at 23° C.in a moist chamber. The digested DNA on the surface are next washed withphosphate buffered saline (PBS) pH 8.0 to remove excess reagent andreaction products. An aliquot of 1 μL of 3-hydroxypicolinic acid isadded to the surface and analyzed again by laser desorptiontime-of-flight mass spectrometry. A nascent sequence of the DNA isgenerated. The sequence is deduced by the differences in the massbetween two peaks.

[0231] Carbohydrate chains are oxidized by periodate and activated to bespecifically coupled to a photolytic compound on a surface. Sequencingof the tethered carbohydrate with specific enzymes such asneuraminidase, mannase, fucase, galactosidase, glucosidase, O— orN-glycanase is carried out and determined by laser desorptiontime-of-flight mass spectrometry. 5-(4-aminophenylazo)salicylic acid(Aldrich) is coupled to a carboxyl reactive surface such as arylamine oncontrolled pore glass beads. The carbohydrate moieties of human andbovine histidine rich glycoprotein are oxidized by low concentration(0.2 M) of sodium meta-periodate in water at 23° C. for 90 min. Theexcess reagents are washed away with water. Add the proteins to the5-(4-aminophenylazo)salicylic acid coupled to controlled pore glassbeads in phosphate buffer, pH 8.0. Then add sodium cyanoborohydride (0.6mg/100 μl) and stir in a fume hood at 23° C. for 18 hr. Wash extensivelywith water, 1 M NaCl, and then water again to remove excess reagents andunreacted proteins. An aliquot of 1 μL of the beads is then deposited onthe probe tip, 1 μL of sinapinic acid (dissolved in 50% methanol/0.1%trifluoroacetic acid) is mixed with the beads and the sample is analyzedby laser desorption time-of-flight mass spectrometry. The remainingproteins bound on the surface are washed free of sinapinic acid withmethanol and incubated with 2 μl of trypsin in phosphate buffer pH 8.0at 37° C. for 30 min. The surface with bound glycopeptides is washedthoroughly with phosphate buffered saline and water to remove excessreagent and unbound peptides. An aliquot of 1 μL of sinapinic acid ismixed with the beads and the sample is analyzed by laser desorptiontime-of-flight mass spectrometry. After obtaining several mass spectra(each an average of 50 laser shots), the remaining glycopeptides on theprobe surface are washed free of sinapinic acid with methanol anddigested in sequence or in combination with N-acetylneuraminidase(NANase III, Glyko, 50 mM sodium phosphate buffer, pH 6.0, 37° C. 1 hr),mannosidase (MANase I, Glyko, 50 mM sodium phosphate, pH 6.0, 37° C. 18hr), fucosidase (FUCase I, Glyko, 50 mM sodium phosphate, pH 5.0, 37° C.18 hr), N-acetylglucosaminidase (HEXase I, Glyko, 50 mM sodiumphosphate, pH 5.0, 37° C. 4 hr), O-glycosidase (Glyko, 50 mM sodiumphosphate, pH 5.0, 37° C. 18 hr) or N-glycanase (PNGase F, Glyko, 100 mMsodium phosphate, pH 8.6, 37° C., 18 hr). The fragmented glycopeptideson the surface are finally washed with phosphate buffered saline andwater to remove the reagents and reaction products. An aliquot of 1 μLof sinapinic acid is added to the surface and analyzed again by laserdesorption time-of-flight mass spectrometry. Nascent sequences of theglycopeptides are observed. The sequences are deduced by the differencesin the mass between two peaks.

[0232] All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

[0233] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Theoligonucleotides, compounds, methods, procedures and techniquesdescribed herein are presently representative of the preferredembodiments, are intended to be exemplary and are not intended aslimitations on the scope. Changes therein and other uses will occur tothose skilled in the art which are encompassed within the spirit of theinvention and are defined by the scope of the appended claims.

1-73. (canceled)
 74. A probe that is removably insertable into a massspectrometer, the probe having a surface for presenting an analyte to anenergy source that emits energy capable of desorbing the analyte fromthe probe for analyte detection, wherein the probe comprises animmobilized photolabile attachment molecule on the probe surface, thephotolabile attachment molecule comprising a photolabile bond and abinding site for binding with the analyte, wherein the analyte isreleasable from the probe surface by cleavage of the photolabile bond.75. The probe of claim 74 wherein the photolabile attachment molecule isimmobilized by covalent binding to the probe surface.
 76. The probe ofclaim 74 wherein the probe surface comprises metal, metal coated with asynthetic polymer, glass, ceramic, a synthetic polymer or a mixturethereof.
 77. The probe of claim 76 wherein the probe surface comprisesglass.
 78. The probe of claim 76 wherein the probe surface comprisesceramic.
 79. The probe of claim 76 wherein the probe surface comprises asynthetic polymer.
 80. The probe of claim 74 wherein the photolabileattachment molecule is immobilized by covalent binding to a solid phaseplaced on the probe surface.
 81. The probe of claim 80 wherein the solidphase comprises a material selected from the group consisting of anelectrically insulating material, a magnetic or paramagnetic material, aflexible material, an optically transparent material, a porous ornon-porous cross-linked polymer and a biopolymer.
 82. The probe of claim81 wherein the material is a bead.
 83. The probe of claim 81 wherein thematerial comprises a porous or non-porous bead of cross-linked polymer.84. The probe of claim 81 wherein the material is selected from thegroup consisting of agarose, dextran and cellulose.
 85. The probe ofclaim 81 wherein the material is glass or a synthetic polymer selectedfrom polystyrene, polypropylene, polyethylene and polycarbonate.
 86. Theprobe of claim 81 wherein the material comprises a magnetic orparamagnetic material.
 87. The probe of any of claims 74, 75 or 80further comprising the analyte bound to the photolabile attachmentmolecule.
 88. The probe of claim 74 wherein the analyte comprises apeptide or protein.
 89. The probe of claim 74 wherein the analytecomprises a carbohydrate.
 90. The probe of claim 74 wherein the analytecomprises RNA or DNA.
 91. The probe of claim 87 wherein the probesurface further comprises a matrix.
 92. The probe of claim 88 whereinthe probe surface further comprises a matrix.
 93. The probe of claim 89wherein the probe surface further comprises a matrix.
 94. The probe ofclaim 90 wherein the probe surface further comprises a matrix.
 95. Theprobe of claim 74 wherein the binding site for binding with the analyteis photolabile.
 96. The probe of claim 74 wherein the binding site forbinding the analyte is available for covalent binding with the analyte.97. The probe of claim 74 wherein the photolabile bond is at a positionother than the binding site for the analyte.
 98. The probe of claim 74wherein the photolabile attachment molecule is5-(4-aminophenylazo)salicylic acid or azodicarbonamide.
 99. The probe ofclaim 74 wherein the binding site for binding with the analyte comprisesan amine group, a hydroxyl group, a sulfhydryl group, a carbonyl groupor an active hydrogen reactive group.
 100. The probe of claim 74 whereinthe photolabile attachment molecule also functions as an energyabsorbing molecule that absorbs energy from the energy source andpromotes desorption of the analyte.
 101. The probe of claim 74 whereinthe binding site comprises an azo group or an arylamine group.
 102. Theprobe of claim 74 wherein the probe surface comprises an array oflocations, each location having at least one analyte deposited thereon.103. A method for desorbing an analyte molecule from a probe surfacecomprising: (a) providing a probe that is removably insertable into amass spectrometer, the probe having a surface for presenting an analyteto an energy source that emits energy capable of desorbing the analytefrom the probe for analyte detection, wherein the probe comprises animmobilized photolabile attachment molecule on the probe surface, thephotolabile attachment molecule comprising a photolabile bond and abinding site to which the analyte is bound, wherein the analyte isreleasable from the probe surface by cleavage of the photolabile bond;and (b) desorbing the analyte molecule from the probe by (1) exposingthe analyte to light from the light source, thereby releasing theanalyte from the probe by photolytic cleavage; and (2) exposing theanalyte to energy from the energy source.
 104. The method of claim 103wherein the photolabile attachment molecule is immobilized by covalentbinding to the probe surface.
 105. The method of claim 103 wherein thephotolabile attachment molecule is immobilized by covalent binding to asolid phase placed on the probe surface.
 106. The method of claim 103wherein the analyte comprises a protein or peptide.
 107. The method ofclaim 103 wherein the analyte comprises a nucleic acid.
 108. The methodof claim 103 wherein the analyte comprises a carbohydrate.
 109. Themethod of claim 103 further comprising before step (b) the step ofmodifying the analyte chemically or enzymatically while bound to thephotolabile attachment molecule.
 110. The method of claim 103 furthercomprising after step (b) the steps of: c) modifying the analytechemically or enzymatically while bound to the photolabile attachmentmolecule; and d) repeating step (b).
 111. The method of claim 103wherein the energy source emits laser light that ionizes the analyte toproduce an ion.
 112. The method of claim 105 further comprisingmodifying the analyte chemically or enzymatically while bound to thesolid phase.
 113. The method of claim 107 wherein the nucleic acid isDNA.
 114. A system for detecting an analyte comprising: a removablyinsertable probe having a surface for presenting an analyte to an energysource that emits energy capable of desorbing the analyte from the probefor analyte detection, wherein the probe comprises an immobilizedphotolabile attachment molecule on the probe surface, the photolabileattachment molecule comprising a photolabile bond and a binding site towhich the analyte is bound, wherein the analyte is releasable from theprobe surface by cleavage of the photolabile bond; a light source thatdirects light to the probe surface causing the photolytic cleavage; anenergy source that directs light to the probe surface, the energydesorbing the analyte from the probe surface; and a detector incommunication with the probe surface that detects the desorbed analyte.115. The system of claim 114 which is a laser desorption massspectrometer wherein: the energy source emits laser light that ionizesthe analyte to produce an ion, the system further comprises means foraccelerating the ion to the detector, the detector detects the ion, andthe system further comprises means for determining the mass of the ion.116. The system of claim 114 wherein the detector detects ions.
 117. Thesystem of claim 114 wherein the detector detects radioactivity or light.118. The system of claim 115 wherein the photolabile attachment moleculeis immobilized by covalent binding to the probe surface.
 119. The systemof claim 115 wherein the photolabile attachment molecule is immobilizedby covalent binding to a solid phase placed on the probe surface. 120.The system of claim 115 wherein the binding site is photolabile. 121.The system of claim 115 wherein the binding site is available forcovalent binding with the analyte.
 122. The system of claim 115 whereinthe photolabile attachment molecule comprises a photolytic chemical bondat a position other than the binding site.
 123. The system of claim 115wherein the photolabile attachment molecule is5-(4-aminophenylazo)salicylic acid or azodicarbonamide.
 124. The systemof claim 115 wherein the binding site comprises an amine group, ahydroxyl group, a sulfhydryl group, a carbonyl group or an activehydrogen reactive group.
 125. The system of claim 115 wherein thephotolabile attachment molecule is photolyzed with the energy source.126. The system of claim 115 wherein the photolabile attachment moleculeis photolyzed with energy different than the energy source.
 127. Thesystem of claim 115 wherein the photolabile attachment molecule alsofunctions as an energy absorbing molecule that promotes desorption ofthe analyte when presented to the energy source.
 128. The system ofclaim 115 wherein the binding site comprises an azo group or anarylamine group.
 129. The system of claim 115 wherein the analytecomprises a protein or peptide.
 130. The system of claim 115 wherein theanalyte comprises a nucleic acid.
 131. The system of claim 115 whereinthe analyte comprises a carbohydrate.